Broad range gene and genotype papillomavirus transcriptome as a biomarker of papillomavirus-associated cancer stages

SEQUENCE LISTING

The ASCII copy of the sequence listing submitted herewith is named Listing_15319893.txt, was created on Sep. 3, 2020, is 334,075 bytes in size, and is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

HPV infections are associated with the development of cervix carcinoma and possibly other cancers like head and neck cancers. For example, cancer of the cervix is one of the most common cancers among women in all countries. Human papillomaviruses (HPV) are the etiologic agents responsible for over 99% of all cancers of the cervix. HPVs are DNA viruses commonly transmitted through sexual contact, which include more than 100 genotypes. Human Papillomaviruses are small, non-enveloped DNA viruses, approximately 55 nm in diameter, that infect basal cells and replicate in the nucleus of squamous epithelial cells. The genomic organization of each of the papillomaviruses is similar and can be divided into three functional regions. Following infection, the early HPV genes (E6, E7, E1, E2, E4 and E5) are expressed and the viral DNA replicates from the episomal form of the virus. In the upper layer of the epithelium the viral genome is replicated further, and the late genes (L1 and L2) and E4 are expressed. The shed virus can then initiate new infections.

Human papillomaviruses (HPV) are viruses displaying a high genetic diversity. About one hundred HPV types which are classified in different genus, mainly the alpha, beta and gamma genus. Within these genus, many species have been identified. HPV classification is based on the genomic sequence of the L1 gene which encodes the major capsid protein. The different HPV types are characterized by their tissue tropism, and HPV types with either cutaneous or mucosal tropism can be distinguished. They are also characterized by their oncogenic potential and one can distinguish between highly oncogenic HPV types (high-risk HPV) and weakly oncogenic HPV types (low-risk HPV).

HPV infections are very common and depending on the HPV types and host immune defense, the infection disappears in 6-12 months in 90% of women. According to a recent CDC report, there are 14 million new HPV infections each year in the USA alone, which account for 50% of sexually transmitted infections (STIs). This means that 1.4 million individuals are each year at risk of developing HPV induced cancer. Two HPV vaccines have been approved, but they are not broadly used in the total population. In addition, these vaccines only cover several types such as HPV6, 11, 16 and 18 and leave unprotected a significant part of the population.

Since HPV are common viruses that can cause usually warts and because there are more than 100 types of HPV, diagnosis and disease management are complex. It is even further complicated taken that most HPVs are deemed harmless, and so far only about 14 types have been shown to be associated with increased risk of cancer. These HPV types affect the genital tract and are transmitted through sexual contact with an infected partner. As of today, HPV types have been classified as low-risk or high-risk HPVs according to observations in clinical cohorts. Low-risk HPVs have been classified according to their association with genital warts; whereas High-risk HPVs (HR HPVs) are identified as a limited number of types which are shown to induce cancers of the cervix, vulva, vagina, and anus in women. In men, these High-risk HPVs can lead to cancers of the anus and penis.

Cancer biomarkers in HPV-related cancers are greatly needed for a better diagnostic of pre-cancer and cancer stages of the disease, prognosis and therapeutic management.

Despite the responsibility of HR HPVs in most cervix cancers, screening tests of cancer remain mainly based on the Pap cytology test and not on HPV tests. This is largely due to the limitations of current molecular tests. HPV DNA identification of HR HPVs is not fully predictive of cancer: only high loads of HPV16 and possibility persistence for months of HR-HPVs are associated with an increased risk of cancer development. Thus, the usage of DNA HPV tests, as a screening assay, shows low positive predictive value for CIN2/3 lesions. Expression of E6 and E7 mRNAs of HR HPVs has been proposed as a better marker of cancer development, but E6 and E7 are expressed during HPV acute infection, so it remains difficult to define a threshold of expression associated to persistence and cancer development.

Low-grade intraepithelial lesions are a site of productive viral replication. Progression to high-grade intraepithelial lesions and invasive carcinomas is associated with a persistent high-risk HPV infection and often integration of the HPV genome into the host chromosomes, loss or disruption of E2 and subsequent upregulation of E6 and E7 expression. E6 and E7 are the oncogenes of the virus and expression of these genes is required for malignant transformation. Among others, E6 and E7 mediate degradation of the tumor suppressors p53 and RB, respectively, and interfere with cell-cycle regulation. E6 and E7 proteins from low-risk types are less competent in interfering with p53 and pRb functions than E6/E7 proteins from high-risk types. Therefore, low-risk HPV infections are associated with benign proliferations, such as genital warts and low-grade intraepithelial lesions prone to regress.

Different techniques are available today for detecting HPV based on DNA typing. For example, the COBAS (Roche) and APTIMA (GEN-PROBE) kits are PCR tests of specific targets intended for the qualitative in vitro detection of mRNA of the L1 gene from 14 types of human papillomavirus (HPV) virus considered High risk (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). However, both of these HPV Assays do not distinguish the differences between the 14 High-risk types. In fact, only 6 different results are obtainable: HPV16 positive or negative, HPV18 positive or negative, others 12 HPVs positive or negative. LINEAR ARRAY HPV Genotyping Test (Roche) is a qualitative test that detects 37 high- and low-risk human papillomavirus genotypes, including those considered a significant risk factor for High-grade Squamous Intraepithelial (HSIL) progression to cervical cancer. This test is a qualitative in vitro test for the detection of Human Papilloma Virus in clinical specimens. The test utilizes amplification of target DNAs by PCR of the late gene L1 of HPV DNA genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (MM9) (novel type related to HPV73), 81, 82 (MM4) (novel type related to HPV82), 83 (MM7) (novel type related to HPV83), 84 (MM8) (novel type related to HPV84), 1539 and CP6108. The digene HC2 HPV DNA Test, developed by Qiagen, is based on Capture Hybridization of HPV DNAs (L1 gene) for the qualitative detection of 18 types (HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 [68a], 73, 82MM4 [821S39]) in cervical specimens.

More recently, NucliSENS EasyQ HPV was made available to qualitative detection of oncogenes E6/E7 mRNAs of 5 specific High risk HPVs 16, 18, 31, 33 and 45. Detection of HPV E6 and E7 has been proposed as a better correlate of cancer development than HPV DNA.

In addition, WO2011/088573 (Her Majesty The Queen In Right of Canada as represented by The Minister of Health), describes a set of probes to detect and Identify 46 specifically targeted species of mucosal human papillomaviruses (HPV). These probes are used as a multiplex assay based on nested PCR amplification and the Luminex xMAP technology for genotyping DNA of L1 genes of HPV types 6, 11, 13, 16, 18, 26, 30, 31, 32, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91 and 97.

All the molecular tests currently described for HPV identification use molecular techniques based on species/genus genotype specific oligonucleotides binding to specifically amplify and/or probe papillomavirus nucleic acids. In addition, these tests all share a specific qualitative detection restricted to some specific HPVs, but not a general and broad range of HPVs. Finally, the presence of HPV nucleic acid, especially DNA, does not mean the presence of cervical dysplasia or cervical cancer.

The inventors analyzed clinical data of cohorts of HPV infected individuals and considered that about 15% of patients are not positive for one of the classified High-risk HPVs but yet, they are at risk of developing HPV induced cancers. Therefore, there is a need to design a new HPV assay with a transversal and broad approach not limited to few specific HPV types. Moreover, the profile expression of the viral genes must be characterized. In fact, HPV infection during cancer development is associated with a shift from productive infection towards non-productive infection characterized by a high level of E6 and E7 and low levels of expression E2 and of late genes, often associated with an integration of HPV DNA in the host chromosomes, at least for HPV16. In connection with the present invention, the inventors investigated shared homology of sequences of all HPV types and no clear global homologies are found within features between polypeptide or nucleic acid sequences.

The inventors designed consensus primers within all intra subgroup alpha, and HPV-species specific composition of primers encompassing splice junctions, genomic and unspliced regions, and human fusion transcript regions of each HPV transcript. It is therefore provided hereafter several compositions of primers depending on the desired specificity and coverage of the test. The inventors propose here an approach for detecting cancer or risk of developing cancer not limited to the specific HPVs known today to be classified as High-risk. Thus, to bypass the limitation of the current tests above, the present invention does not rely on the above 14 HR species/genotype specific oligonucleotides binding to specifically amplify and/or probe papillomavirus few DNAs or few transcripts. It relies in one aspect on identification of the different gene transcripts based on High-Throughput sequencing, which allows for further transcripts/species/genotype identification based on sequence comparison with known alpha-papillomavirus sequences in relevant databases. This has the advantage of being capable of testing simultaneously the different transcripts of a great number of papillomavirus genotypes/species and to identify relevant biomarkers along the wide range of HR and LR HPVs.

The invention also provides generic consensus primers allowing a broad amplification or pre-amplification of relevant genes of alpha HPV, not depending on the specific transcripts of the 14 HR HPVs, which are amenable to PCR testing or enhance signal/noise ratio in connection with the High-Throughput sequencing mentioned above.

More particularly, the invention relates to identification of all E6/E7 transcripts in a given sample, and recognizing to which species/genotypes they belong to, sorting the reads corresponding to other viral transcripts of the same genotype/species so as to compute ratios defining relative molecular abundance of transcripts within this (these) given genotype(s) as biomarker of cancer development.

The inventors therefore designed a kit for HPV diagnosis based on a broad screening of the level of E6 and/or E7 mRNAs within the group alpha of HPVs. In the test described in details below, the inventors designed consensus primers allowing amplification or pre-amplification E6 and/or E7 mRNAs of genotypes of sub-group alpha to detect the level of expression of, wherein a significant expression level of E6 and/or E7 of group alpha HPVs in a single time point or over time is indicative of risk of developing HPV induced cancers. Following broad range amplification with consensus HPV primers or with HPV primers designed to perform a first step of HPV specific Reverse transcription reaction, the inventors also propose to quantitate E6 and or E7 as reads delivered by next generation sequencing techniques.

The present invention also provides a method for determining the level of expression of structural or late viral proteins such as L1 or L2. In such embodiment, a ratio R1 between E6 and/or E7 and L1 and/or L2 is determined, and compared to a ratio R found in low risk or non-persistent HPV infections, wherein a ratio R1 below a reference value R is indicative of HPV infections associated with higher risk of developing genital neoplasia and cancer.

GENERAL DESCRIPTION OF THE INVENTION

The invention provides a high through put sequencing method allowing relative quantification of reads across oncogenic viruses, such as polyoma virus or group alpha HPV, preferably group alpha HPV, comprising enrichment of the viral RNAs in a sample using random or consensus pre-amplification and/or specific reverse transcriptase reaction, determining the number of reads matching said viruses based on species discrimination, comparing the most prevalent high risk species, further determining within said most prevalent high risk species the relative number of reads matching at least one oncogenic gene, preferably two oncogenic genes, compared to at least one non oncogenic gene, preferably several non oncogenic genes. From these discrimination steps on species and interspecies read numbers, ratios are calculated to detect increase in relative level of high risk species versus low risk species and ratios within said high risk species of reads matching oncogenic genes versus structural or regulatory genes. Applied to HPV, this test encompasses determining the level of HR HPVs reads versus LR HPVs reads through group alpha HPVs, determining the ratio of early versus late genes (E6 and or E7 versus L1 and/or L2) within the most prevalent HR HPVs, and assessing risk of developing HPV induced cancer in patients which said ratio tend towards infinity. Refined ratios can be obtained using a filter applied on reads mapping specifically RNA spliced events.

In one embodiment, the method is for assessing risk of developing HPV induced cancer in patients infected with at least two different HPV species of group alpha.

In one embodiment, the method is for assessing HPV virus clearance in patient receiving HPV preventive or curative HPV vaccine.

In a first aspect, the present invention relates to a method for determining a patient risk of developing oncogenic virus induced cancer, such as polyoma virus or group alpha HPV comprising:

a) enrichment of the viral RNAs in a sample using random or consensus pre-amplification and/or specific reverse transcriptase reaction, preferably consensus pre-amplification;
b) sequencing cDNA produced in step a), and generating reads of said cDNA;
c) determining the number of reads matching said viruses based on species discrimination and determining the most prevalent high risk species present in the sample relative to other species;
d) determining within said most prevalent high risk species the relative number of reads matching least one oncogenic gene compared to at least one non oncogenic gene, preferably oncogenic genes compared to non oncogenic genes;
e) computing ratios within said high risk species of reads matching at least one oncogenic gene versus corresponding interspecies structural or regulatory gene, preferably oncogenic genes versus corresponding interspecies structural or regulatory genes;
f) determining risk of developing oncogenic virus induced cancer in patients wherein said ratio tend towards infinity, such as for example when ratio R is between 0.25, 0.4, 0.5, 1 to infinity.

This method is suitable for diagnosis or prognosis of risk to develop virus induced cancer in a human subject.

This method is particularly suited for assessing risk of developing HPV induced cancer in patients infected with at least two different HPV species of group alpha, for example multiple infected with HPV16, HPV35 and HPV6.

This method is also specifically suitable for assessing HPV virus clearance in patient receiving HPV preventive or curative HPV vaccine. It can be performed before vaccination to confirm vaccine potential for clearing existing infections or post-vaccination for follow-up.

In a preferred embodiment of step a), the enrichment of the viral RNAs is performed by a reverse transcription of the viral RNAs, and an amplification of the produced cDNA by multiplex-PCR with a group alpha HPV-specific composition of primers encompassing splice junctions, genomic and unspliced regions, and human fusion transcript regions of each HPV transcript.

In another embodiment, the reverse transcription is performed with random hexamers.

In another embodiment, the reverse transcription is performed with HPV-specific primers.

In another embodiment, the reverse transcription (RT) and the multiplex amplification are performed in the same tube (one-step RT-PCR).

In another preferred embodiment of step a) above, consensus pre-amplification comprises random reverse transcription of the viral RNAs followed by a multiplex amplification of the HPV transcripts.

Advantageously, the random reverse transcription is performed with random hexamers.

Advantageously, the multiplex amplification of the HPV transcripts is performed with HPV-specific primers.

In a preferred embodiment of step b) above, the sequencing is a High throughput sequencing method.

Ratio (R) is calculated as the number of reads of at least one early HPV16 transcript to the number of reads of at least one late HPV16 transcript, with a higher ratio (R) correlating with an increased risk of developing high-grade malignant HPV-induced cancer. This method further include correlating a higher number of reads of HPV16 transcripts relative to reads of transcripts of another HPV species with an increased risk of developing high-grade malignant HPV-induced cancer. To obtain sufficient number of reads, the cDNA is generated using random primers or using HPV-specific primers.

For example, the ratio is calculated by calculating a ratio (R1) of the number of reads of one HPV HR E6 and/or E7 transcripts to the number of reads of said one HPV HR L1 and/or L2 transcripts and the ratio is calculated by calculating a ratio (R2) of the number of reads of a second HPV HR E6 and E7 transcripts to the number of reads of said second HPV HR L1 and L2 transcripts. This method is applicable to determining the number of HPV sequence reads of at least 2 Alpha group HPV species, including for example HPV16.

In a specific embodiment but applied to oncogenic viruses in general such as polyoma or HPV, the method of the invention comprises:

- a) optionally, pretreating nucleic acids to remove human genomic DNA,
- b) optionally, pre-amplify viral mRNAs, wherein said viral mRNAs comprises oncogenic mRNAs and at least one other mRNA,
- c) sequencing mRNAs, or cDNAs thereof, obtained after steps a) and b), in the sample of a human subject,
- d) identifying the reads corresponding to said oncogenic mRNAs,
- e) identifying to which species or genotypes said oncogenic mRNAs of step d) belong to,
- f) sorting the reads corresponding to said at least one other viral mRNAs, or cDNAs thereof obtained after steps a) and b), of the same genotype or species identified in step e),
- g) optionally, identifying fusion transcripts as a signature of viral DNA integrations events in the host chromosome and/or additional human cancer cell biomarkers,
- h) optionally, deleting all other sequences including human sequences which are not sequences identified and sorted following steps d), e), f) and g),
- i) computing ratios R defining molecular abundance of said oncogenic mRNAs relative to said at least one other viral mRNAs of the same genotype or species of step f), wherein an increase of ratios R correlate with an increased risk of developing viral induced cancer.

By virus induced cancer, it is more particularly contemplated herein Papova virus induced cancer, more specifically Papilloma or Polyoma virus induced cancer, preferably Papilloma virus induced cancer.

By other viral mRNAs in step f) it is meant mRNAs of viral genes selected from structural genes, for example capsid genes as well as from regulatory genes, and replication/transcription genes.

In a particular embodiment, the present invention relates to a method for diagnosis or prognosis of risk to develop HPV induced cancer in a human subject comprising:

- a) optionally, pretreating nucleic acids to remove human genomic DNA,
- b) optionally, pre-amplify HPVs mRNAs, wherein said mRNAs comprises E6 and/or E7 HPV RNAs and at least one other HPV mRNAs,
- c) sequencing nucleic acids in the sample of a human subject or obtain after steps a) and b),
- d) identifying the reads corresponding to E6 and/or E7 HPV RNAs,
- e) identifying to which species or genotypes E6 and/or E7 HPV mRNAs of step d) belong to,
- f) sorting the reads corresponding to other viral HPV mRNAs of the same genotype or species identified in step e),
- g) optionally, identifying fusion transcripts as a signature of HPV DNA integrations events in the host chromosome and/or additional human cancer cell biomarkers,
- h) optionally, deleting all other sequences including human sequences which are not sequences identified and sorted following steps d), e), f) and g),
- i) computing ratios defining molecular abundance of E6 and/or E7 HPV mRNAs relative to said other viral transcripts of the same genotype or species of step f),
  
  wherein an increased level of said ratios correlates with an increased risk of developing viral induced cancer.

By other viral mRNAs in step f), it is more particularly referred to selected mRNAs from genes coding for capsid proteins (L1 and L2), gene coding for the growth stimulation protein (E5), genes coding for replication or transcription proteins (E4, E2 and E1, E8). In step g), additional human cancer cell biomarkers can be selected for example from PRC1, CCNB2, SYCP2 CDKN3, NUSAP1, CDC20, p16INK4a, Ki-67.

In one specific embodiment, step f) comprises sorting the reads of L1 and/or L2 HPV mRNAs corresponding to the species or genotype of E6 and/or E7 HPV mRNAs identified in step d). In this embodiment, step h) comprises computing ratios defining relative molecular abundance of E6 and/or E7 HPV mRNAs relative to the reads of L1 and/or at least one other viral mRNAs corresponding to the species or genotype of E6 and/or E7 HPV mRNAs. In such embodiment, step b) optionally comprises pre-amplifying HPVs mRNAs, wherein said mRNAs comprises E6 and/or E7 HPV RNAs and L1 and/or at least one other viral HPV mRNAs.

In a second aspect, the present invention relates to a method for diagnosis risk to develop HPV induced cancer comprising:

(a) determining the level of at least a first marker selected from E6 mRNAs of group alpha HPVs, E7 mRNAs of group alpha HPVs, or both, in the sample of a patient or in the sample of an individual suspected to be infected by HPV,
(b) comparing the levels determined in step (a) to a reference value of E6 mRNAs of group alpha HPVs, E7 mRNAs of group alpha HPVs, or both in low risk individuals infected with HPVs,
(c) wherein an increased level as determined in step a) compared to the reference level in step b) is indicative of higher risk to develop HPV cancer induced.

It must be contemplated that these biomarkers are not restricted to E6 or E7 mRNAs of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 but extend to all E6 or E7 mRNAs of HPVs of several genus alpha, comprising and covering HPVs of groups α5, 6, 7, 10; optionally extending to additional alpha group HPVs as desired.

In this second aspect, the levels of E6 mRNAs of group alpha HPVs, E7 mRNAs of group alpha HPVs, or both are determined inter alia by hybridization with a labeled probe, amplification, including PCR, nucleic acid microarrays, high-throughput sequencing with or without pre-amplification. The measure may be carried out directly on an extracted messenger RNA (mRNA) sample, or on reverse transcribed complementary DNA (cDNA) prepared from extracted mRNA. From the mRNA or cDNA sample, the amount of nucleic acid transcripts is determined using nucleic acid microarrays, quantitative PCR, hybridization with a labeled probe, or directly by counting corresponding reads following high-throughput sequencing.

For both first and second aspect, amplification or pre-amplification is depicted in details below with specifically designed consensus primers allowing generic pre-amplification of all or desired HPVs belonging to group alpha, in particular pre-amplification of the specific domains of the group alpha, preferably of the HR-αHPV. According to the invention, primers depicted below are provided to amplify and detect the amount of E6 mRNAs and E7 mRNAs of all or several group alpha HPVs depending on the desired scope of the test. Therefore, the invention provides a much broader test extending beyond types such as HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 which are today classified as High-risk HPVs; as it allows the determination of high level expression of E6 and/E7 of several alpha subgroups of HPVs and even covering the all HPVs of the alpha group.

For example, In the above method, the quantification is performed on E6 and/or E7 mRNAs of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58 and papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82. The combined generic consensus primers to generically amplify alpha subgroups are described herein-after.

In a specific embodiment, the method of the invention further refines the above method aiming at the quantitative determination of expression levels of a panel of biomarkers in biological samples of patients or individuals suspected to be infected with HPVs, which combined biomarkers are indicative and/or predictive, in a single time-point, of patients at risk of developing HPV induced cancers.

The panel provided by the invention comprises the following biomarkers:

- At least a first marker selected from E6 mRNAs of group alpha HPVs, E7 mRNAs of group alpha HPVs, or both,
- At least a second marker selected from L1 mRNAs of group alpha HPVs, L2 mRNAs of group alpha HPVs, or both,
- wherein said E6, E7, L1 and L2 mRNAs have corresponding intragenetic sequences,
- optionally, at least one host cellular marker indicative of neoplasia or cancer.

For example, the panel is composed of at least 5, 10, 20, 30 or 50 different mRNAs of said E6, E7, L1 and L2 mRNAs of group alpha HPVs. In one specific embodiment, the panel is composed of all group alpha HPVs mRNAs of E6 and/or E7, and L1 and/or L2. The combined generic consensus primers to generically amplify alpha subgroups depending on the desired scope of the test are provided herein-after. Alternatively, the invention is performed using sequence-independent-amplified or direct HT-sequencing for quantitative detection of individual mRNA of E6 and/or E7 HPV sequences belonging to the HPV alpha group.

Such method according to the invention is also provided for predicting the progression of HPV infection in a patient suffering from HPV infection.

In one embodiment, the invention encompasses a method for assessing a human papilloma virus (HPV) infected patient comprising: generating cDNA from a patient sample comprising RNA; sequencing the cDNA; generating reads of sequence of the cDNA; discriminating HPV sequence reads on the basis of HPV specie; discriminating HPV sequence reads on the basis of HPV gene transcript; quantitating the number of HPV sequence reads according to HPV species and HPV gene transcript, determining the number of HPV sequence reads of at least 2 HPV gene transcripts; and determining the number of HPV sequence reads of at least 2 HPV species; wherein the patient sample contains 2 or more HPV species.

In a further embodiment, the method comprises calculating a ratio (R) of the number of reads of at least one early HPV16 transcript to the number of reads of at least one late HPV16 transcript, with a higher ratio (R) correlating with an increased risk of developing high-grade malignant HPV-induced cancer.

In another embodiment, the method comprises correlating a higher number of reads of HPV16 transcripts relative to reads of transcripts of another HPV species with an increased risk of developing high-grade malignant HPV-induced cancer.

In one embodiment, the cDNA is generated using random primers. In one embodiment, the cDNA is generated using HPV-specific primers (i.e., primers specific to domains of a HPV, such as a HR-αHPV, comprising splice junctions, genomic and unspliced regions, and human fusion transcript regions of each HPV transcript).

In one embodiment, the ratio is calculated by calculating the ratio (R) of the number of reads of HPV16 E6 and/or E7 transcripts to the number of reads of HPV16 L1 and/or L2 transcripts. In one embodiment, the ratio is calculated by calculating the ratio (R) of the number of reads of HPV16 E6 and E7 transcripts to the number of reads of L1 and L2 transcripts.

In one embodiment, the method comprises determining the number of HPV sequence reads of at least 2 alpha group HPV species. In one embodiment, the method comprises generating at least 10⁶reads of sequence of the cDNA. In one embodiment, the method comprises generating at least 10⁷reads of sequence of the cDNA.

In one embodiment, the invention encompasses a method for assessing a human papilloma virus (HPV) infected patient comprising generating cDNA from a patient sample comprising RNA; sequencing the cDNA; generating reads of sequence of the cDNA; discriminating HPV sequence reads on the basis of HPV gene transcript; quantitating the level of HPV sequence reads according to HPV gene transcript; determining the number of HPV sequence reads of at least one HPV early gene transcript; determining the number of HPV sequence reads of at least one HPV late gene transcript; and determining the ratio of the number of HPV sequence reads of at least one HPV early gene transcript to the number of HPV sequence reads of at least one HPV late gene transcript.

In one embodiment, the method comprises calculating a ratio (R) of the number of reads of at least one early HPV16 transcript to the number of reads of at least one late HPV16 transcript, with a higher ratio (R) correlating with an increased risk of developing high-grade malignant HPV-induced cancer.

In one embodiment, the at least one early transcript is HPV E6 or E7 and the at least one late transcript is L1 or L2. In one embodiment, the at least one early transcript is HPV E6 and E7 and the at least one late transcript is L1 and L2.

In one embodiment, the cDNA is generated using random primers. In one embodiment, the cDNA is generated using HPV specific primers.

Some of the terms used throughout the specification are specifically defined here below:

Definitions

Biological samples as referred herein include, without limitation, mammalian bodily fluids, especially oral fluids or scrapings, genital scrapings, in particular cervix scrapings.

HPV alpha group: HPVs are contained within five evolutionary groups. HPV types that infect the cervix come from the Alpha group which contains over 60 members. HPV types from the Beta, Gamma, Mu and Nu groups or genus primarily infect cutaneous sites. Alpha papillomaviruses can be subdivided into three categories (high risk, low risk and cutaneous), depending on their prevalence in the general population and on the frequency with which they cause cervical cancer. High-risk types come from the Alpha 5, 6, 7, and 10 groups.

Primers encompassed by the invention are not limited to the sequences defined in the primers depicted below but they can comprise extra bases at the 5′ end, for example from 1 to 5 extra bases as extension corresponding to sequences of the corresponding HPVs E6 or E7. Also, primers shall be understood as embracing shorter sequences of at least 12, 15, 20 or 25 consecutive bases of the primers featured below. In some embodiments, it shall be understood that the invention also contemplates generic probes which have the sequences of the primers depicted herein and which are directly or indirectly labeled. The probes and primers can be extended or swifted from 1 to 15 bases depending on the desired specificity of the PCR amplification step and/or on the specificity of the detection step using standard parameters such as the nucleic acid size and GC contents, stringent hybridization conditions and temperature reactions. For example, low stringency conditions are used when it is desired to obtain broad positive results on a range of homologous targets whereas high stringency conditions are preferred to obtain positive results only if the specific target nucleic is present in the sample. As used herein, the term “stringent hybridization conditions” refers to conditions under which the primer or probe will hybridize only to that exactly complementary target(s). The hybridization conditions affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are optimized to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+, typically about 0.01 to 1.0 M Na+ concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions include hybridization with a buffer solution of 20-30% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 2*SSC at 40° C. Exemplary high stringency conditions include hybridization in 40-50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1*SSC at 60° C. Determination of particular hybridization conditions relating to a specified nucleic acid is routine and is well known in the art, for instance, as described in J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; and F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002.

Preferred PCR primers, which can be used separately or together as a set to amplify a HPV nucleic acid sequence comprise the primers.

α1:

E6

Forward:

(SEQ ID NO. 1)

5′-RGTACWTCTGCCTCATCACAGCC-3′

Reverse:

(SEQ ID NO. 2)

3′-CTCTGCAMTGSGTACASCGAC-5′

E7

Forward:

(SEQ ID NO. 3)

5′-GGARASRCRCCWACSCTAAAGGA-3′

Reverse:

(SEQ ID NO. 4)

3′-CACGCRGGCACACAAWGGACA-5′

L1

Forward:

(SEQ ID NO. 5)

5′-GCGGCCTAGTGACRACAAGG-3′

Reverse:

(SEQ ID NO. 6)

3′-GCACGYAACCCRGCYTGCAG-5′

α2:

E6

Forward:

(SEQ ID NO. 7)

5′-GHGHGCCMTAYGSTGCCTGTG-3′

Forward:

(SEQ ID NO. 8)

5′-CKCCSTACGGTGCWTGTGC-3′

Reverse:

(SEQ ID NO. 9)

3′-GCGGACCGTGCATCKTRWCCA-5′

Reverse:

(SEQ ID NO. 10)

3′-GGCTTTGGCCCATGCATCGT-5′

Reverse:

(SEQ ID NO. 11)

3′-GTGCATCGTGACCAGCAGTAC-5′

E7

Forward:

(SEQ ID NO. 12)

5′-TTGRDTCTTGCACCAGAGGMCGT-3′

Forward:

(SEQ ID NO. 13)

5′-TGCACGGTCCGCATCCCAC-3′

Forward:

(SEQ ID NO. 14)

5′-TGTCTATGGGTGCACAAGAACCC-3′

Reverse:

(SEQ ID NO. 15)

3′-CCCTTATATCTGCKTSGCTGCWS-5′

Reverse:

(SEQ ID NO. 16)

3′-GCAGCGAGGRCACACGASC-5′

Reverse:

(SEQ ID NO. 17)

3′-GGACCGTGCATCGTGACCA-5′

L1

Forward:

(SEQ ID NO. 18)

5′-ATGGCWYTSTGGCGCYCTAGTG-3′

Reverse:

(SEQ ID NO. 19)

3′-CCTCCARGCTAGTRGAYGGYGGY-5′

Reverse:

(SEQ ID NO. 20)

3′-GGGRACYACYGAACGMCGKCGCG-5′

α3:

E6

Forward:

(SEQ ID NO. 21)

5′-AGTGGACRGGRAAGTGCWGCAAC-3′

Forward:

(SEQ ID NO. 22)

5′-YTGTGCAAAGACTGCGASGTGG-3′

Forward:

(SEQ ID NO. 23)

5′-ACTGGCCATTTGGAGTMTGCGC-3′

Reverse:

(SEQ ID NO. 24)

3′-GGCCRYGCATGTTRCYCTACAGT-5′

Reverse:

(SEQ ID NO. 25)

3′-CACYKTCCTGTCCACTBYCCWGC-5′

Reverse:

(SEQ ID NO. 26)

3′-CCAGTGYCGTAGCTCYCGYRYC-5′

Reverse:

(SEQ ID NO. 27)

3′-CTGGCCGTGCATRSYCCTCT-5′

E7

Forward:

(SEQ ID NO. 28)

5′-VAGCAMAGCWGGCCYWTAGGGTG-3′

Forward:

(SEQ ID NO. 29)

5′-KGYWGAACRRGCACAGCAGGCC-3′

Reverse:

(SEQ ID NO. 30)

3′-GGCCACYRCKTCCACYATAAGCT-5′

Reverse:

(SEQ ID NO. 31)

3′-CAGCYGGGACACACTATRTCCAC-5′

Reverse:

(SEQ ID NO. 32)

3′-GCGCAGCSVGGACACACTAT-5′

L1

Forward:

(SEQ ID NO. 33)

5′-CTWTGTGGCGRCMTGGTGAYGGC-3′

Reverse:

(SEQ ID NO. 34)

3′-GGARGGAGGGGGCAMWACMCC-5′

Reverse:

(SEQ ID NO. 35)

3′-CCCTGBGCVCGNTGYAGCCAR-5′

α4:

E6

Forward:

(SEQ ID NO. 36)

5′-SAGTATGGTYTGGAGCTAGAGGA-3′

Reverse:

(SEQ ID NO. 37)

3′-GTCCSGTCCACYGGCCKGM-5′

E7

Forward:

(SEQ ID NO. 38)

5′-MCGMCCCAGCCTSRMGGAC-3′

Reverse:

(SEQ ID NO. 39)

3′-CCTCCATRACGCTABGCGCAG-5′

L1

Forward:

(SEQ ID NO. 40)

5′-TGGCCTAAACGACGTAAACGTGT-3′

Forward:

(SEQ ID NO. 41)

5′-TTCTTTGCAGATGGCTWTGTGGC-3

Reverse:

(SEQ ID NO. 42)

5′-YGTGTCTCGMAARCGCRCCGC-3′::

3′-GCGGYGCGYTTKCGAGACACR-5′

Reverse:

(SEQ ID NO. 43)

5′-CGCAAGTTYTTRYTGCAGCGGGG-3′::

3′-CCCCGCTGCARYAARAACTTGCG-5′

α5:

E6

Forward:

(SEQ ID NO. 44)

5′-GRGAAAGACCACGAACGCTGC-3′

Forward:

(SEQ ID NO. 45)

5′-AATAGCAGGGYASTGGAAAGGGT-3′

Reverse:

(SEQ ID NO. 46)

3′-GCAATTWGCRCAYTGYCCCGTCC-5′

Reverse:

(SEQ ID NO. 47)

3′-TTGTGTTTCTGTTTGGCGCCTTG-5′

Reverse:

(SEQ ID NO. 48)

3′-GCCTTGGTCTCCAGCAGTTTG-5′

E7

Forward:

(SEQ ID NO. 49)

5′-YTAGATYTGGTGCCGCAACCCG-3′

Forward:

(SEQ ID NO. 50)

5′-MGCCATGCGTGGTAATGTACCAC-3′

Reverse:

(SEQ ID NO. 51)

3′-CTCCASCRCTCGRACGTTCTGT-5′

Reverse:

(SEQ ID NO. 52)

3′-CACGGGCAMACCAGGCTTAGK-5′

L1

Forward:

(SEQ ID NO. 53)

5′-KCAGATGGCYTTGYGGCGTACTA-3′

Forward:

(SEQ ID NO. 54)

5′-TGGCYTTGYGGCGTACTAGTGAC-3′

Forward:

(SEQ ID NO. 55)

5′-TGTATTTRCCACCTGCACCWGTG-3′

Reverse:

(SEQ ID NO. 56)

3′-GGGGCRTYRCGYTGACAKGTAGT-5′

Reverse:

(SEQ ID NO. 57)

3′-GGCMGGSCKTTTAAGGCCTGGT-5′

α6:

E6

Forward:

(SEQ ID NO. 58)

5′-GARCGHCCACGWASHBTGCACC-3′

Forward:

(SEQ ID NO. 59)

5′-AATACAGRMGAGCGMCCACGTAC-3′

Forward:

(SEQ ID NO. 60)

5′-RCAATMCACAGGAACGTCCACGA-3′

Reverse:

(SEQ ID NO. 61)

3′-CCTCTGGTGTCAACGGMTGTTGA-5′

Reverse:

(SEQ ID NO. 62)

3′-TCTCCARCACYSCAAACATGACC-5′

E7

Forward:

(SEQ ID NO. 63)

5′-GRACAGCTCAGAGGAWGAGGATG-3′

Forward:

(SEQ ID NO. 64)

5′-GCTCAGAGGAWGAGGATGAGG-3′

Forward:

(SEQ ID NO. 65)

5′-YTRCWGRAGCRGCCACAGCAAGC-3′

Forward:

(SEQ ID NO. 66)

5′-GRAGCRGCCACAGCAAGCTAG-3′

Forward:

(SEQ ID NO. 67)

5′-GAACAGCTCAGAGGAWGAGGATG-3′

Forward:

(SEQ ID NO. 68)

5′-ARTAGACCATTTGCWGGAGCGGC-3′

Reverse:

(SEQ ID NO. 69)

3′-GCCTTGTTGCRCASAGGGG-5′

Reverse:

(SEQ ID NO. 70)

3′-CGCAGAGTGGGCACGTTACT-5′

L1

Forward:

(SEQ ID NO. 71)

5′-TTGCAGATGGCGRYGTGGCG-3′

Reverse:

(SEQ ID NO. 72)

3′-CACCTAAAGGYTGDCCDCGGC-5′

α7:

E6

Forward:

(SEQ ID NO. 73)

5′-TASAGGACAGTGYCGMCRSTGC-3′

Forward:

(SEQ ID NO. 74)

5′-TCMCAAYCCTGMRGAACGGCCAT-3′

Forward:

(SEQ ID NO. 75)

5′-ASAGGACAGTGTCGYSGGTG-3′

Forward:

(SEQ ID NO. 76)

5′-TGCCAGAAACCRTTGAAYCCAGC-3′

Reverse:

(SEQ ID NO. 77)

3′-GTCTGCGGTCCTCYCGBTTDST-5′

Reverse:

(SEQ ID NO. 78)

3′-CTGSCCTCKRTASTGCCCAGCT-5′

Reverse:

(SEQ ID NO. 79)

3′-CACCAGTGTTTCACTACGCGC-5′

Reverse:

(SEQ ID NO. 80)

3′-GCCTTGCTGTTCTTGTGCACG-5′

Reverse:

(SEQ ID NO. 81)

3′-GTCTGGAAAGCCTTTCTTGCCGT-5′

E7

Forward:

(SEQ ID NO. 82)

5′-GACGRGMHGAACMACARCGTCAC-3′

Forward:

(SEQ ID NO. 83)

5′-GACGRGMHGAACMACAGCGTCAC-3′

Forward:

(SEQ ID NO. 84)

5′-ARCACCYTGTCCTTTGTGTGTCC-3′

Reverse:

(SEQ ID NO. 85)

3′-GTGWSTCCATAAACAGCWGCWGT-5′

Reverse:

(SEQ ID NO. 86)

3′-CACACCAMGGACACACAAAGGAC-5′

L1

Forward:

(SEQ ID NO. 87)

5′-GCGBTCTAGYGACARCAHGGTGT-3′

Forward:

(SEQ ID NO. 88)

5′-HCCTGCTATTGGKGARCAYTGGG-3′

Reverse:

(SEQ ID NO. 89)

3′-CCAGTGYTCYCCMATRGCRGGWA-5′

Reverse:

(SEQ ID NO. 90)

3′-TAGASCCACTDGGWGANGGRGAA-5′

α8:

E6

Forward:

(SEQ ID NO. 91)

5′-WATGWCTGCACGKWGCKGCTCC-3′

Reverse:

(SEQ ID NO. 92)

3′-GTAGGCARTATCCYTTCCACRCG-5′

Reverse:

(SEQ ID NO. 93)

3′-CTCCGAGCGTTGGCCTTTC-5′

E7

Forward:

(SEQ ID NO. 94)

5′-GCGTGAGCAAYCCACGCAAC-3′

Reverse:

(SEQ ID NO. 95)

3′-CAGCCATKGYAGTCACACMGCTG-5′

Reverse:

(SEQ ID NO. 96)

3′-TGCCATTGTTGTCACKCTGTAGC-5′

L1

Forward:

(SEQ ID NO. 97)

5′-CCYCCHATKGGNGAATATTGGGG-3′

Reverse:

(SEQ ID NO. 98)

3′-GGAGGATGGTGCWGMACGC-5′

Reverse:

(SEQ ID NO. 99)

3′-GGGTGACTGRCYYAGAAGAGGAA-5′

α9:

E6

Forward:

(SEQ ID NO. 100)

5′-AGTRMARATGCCTCCACGYCTGC-3′

Forward:

(SEQ ID NO. 101)

5′-CTGCACAGGACCAGATGGC-3′

Reverse:

(SEQ ID NO. 102)

3′-TCCATGCATGWTGWCCAGCARTG-5′

Reverse:

(SEQ ID NO. 103)

3′-GCAGCGMCCYTTCCAGGTRTCK-5′

Reverse:

(SEQ ID NO. 104)

3′-GGCATTTCGCCCACCATTGTTAT-5′

E7

Forward:

(SEQ ID NO. 105)

5′-GCYTACACTGCTGGACAACATGC-3′

Forward:

(SEQ ID NO. 106)

5′-AGACAGCTCAGAAGABGAGGTGG-3′

Forward:

(SEQ ID NO. 107)

5′-AACAATGGTGGGCGAAATGCCAG-3′

Reverse:

(SEQ ID NO. 108)

3′-CGTCCGCCATCSTTGTTATGKYT-5′

Reverse:

(SEQ ID NO. 109)

3′-CCTGTRCACTSCACMACMAGCC-5′

Reverse:

(SEQ ID NO. 110)

3′-CTGTCGCTGTAGGGTGCACA-5′

L1

Forward:

(SEQ ID NO. 111)

5′-ATGTGCCTCCTCCYRMCCCWGTA-3′

Forward:

(SEQ ID NO. 112)

5′-AGATGGCTGTCTGGTTACCAGC-3′

Reverse:

(SEQ ID NO. 113)

3′-CCATAWGGRTCYGCAGCCATTTG-5′

Reverse:

(SEQ ID NO. 114)

3′-GCCTTACGCCTGCGCTTGG-5′

α10:

E6

Forward:

(SEQ ID NO. 115)

5′-CCSARSTGTAAWCATGCRTGGAG-3′

Forward:

(SEQ ID NO. 116)

5′-MCGSAMCCTGCACGAATTGTGTG-3′

Forward:

(SEQ ID NO. 117)

5′-CARGACRCWGAGGARAAACCACG-3′

Reverse:

(SEQ ID NO. 118)

3′-CCAACACWCTGAACASCGYCC-5′

Reverse:

(SEQ ID NO. 119)

3′-CCATGCATGATTACASCTSGGTT-5′

Reverse:

(SEQ ID NO. 120)

3′-GTCGGGRYCTCCAACACRCYG-5′

Reverse:

(SEQ ID NO. 121)

3′-CTCCACGCATGTTTACACTTGGG-5′

E7

Forward:

(SEQ ID NO. 122)

5′-GCWCAYTWGGAATHGTGTGCCCC-3′

Forward:

(SEQ ID NO. 123)

5′-CSTGTAAMAACGCCATGAGAGGA-3′

Forward:

(SEQ ID NO. 124)

5′-CGCCATGAGAGGAMACAASCCA-3′

Reverse:

(SEQ ID NO. 125)

3′-GGCACACDATTCCWARTGWGCCC-5′

Reverse:

(SEQ ID NO. 126)

3′-GGTTCGTASGTCRSTTGYTGTAC-5′

Reverse:

(SEQ ID NO. 127)

3′-GTGCACAGSYGGGRCACACWAYT-5′

L1

Forward:

(SEQ ID NO. 128)

5′-GARGCCACWGTSTACYTGCCTC-3′

Forward:

(SEQ ID NO. 129)

5′-ACAGATGTCTCTGTGGCGGC-3′

Reverse:

(SEQ ID NO. 130)

3′-GGATGNCCACTWAYRCCHACDCC-5′

Reverse:

(SEQ ID NO. 131)

3′-GAGGWWACCATAGARCCACTRGG-5′

Reverse:

(SEQ ID NO. 132)

3′-GTGCACGYTGTAGCCAATAWGGC-5′

Reverse:

(SEQ ID NO. 133)

3′-TCCTGTAAACTRGCAGAYGGAGG-5′

Reverse:

(SEQ ID NO. 134)

3′-GGCCYTGTGCWCGTTGYAACCAA-5′

α11:

E6

Forward:

(SEQ ID NO. 135)

5′-GAACGRCCATACAAGCTACMAGC-3′

Reverse:

(SEQ ID NO. 136)

3′-GCAGATGGTCTCCAGCACYG-5′

E7

Forward:

(SEQ ID NO. 137)

5′-WATTGTGTGCCCCAACTGTTCCA-3′

Reverse:

(SEQ ID NO. 138)

3′-CTGGAACAGTTGGGGCACACA-5′

L1

Forward:

(SEQ ID NO. 139)

5′-AGTTCTATCTTCCTCCCCAGCC-3′

Reverse:

(SEQ ID NO. 140)

3′-GGACGKGCACGCATACCWAG-5′

α13:

E6

Forward:

(SEQ ID NO. 141)

5′-TGTCTGCTACTGAACCCCACAC-3′

Reverse:

(SEQ ID NO. 142)

3′-GGCTTCCAGCAATGTAGACACC-5′

E7

Forward:

(SEQ ID NO. 143)

5′-GTTTGACCTGTACTGCAGGGAG-3′

Reverse:

(SEQ ID NO. 144)

3′-GTGAAGCACAGGTGGGACACA-5′

L1

Forward:

(SEQ ID NO. 145)

5′-AAAGTATACCTGCCTCCTACCCC-3′

Reverse:

(SEQ ID NO. 146)

3′-GCACGCTTGCGCGCTGTAC-5′

α14:

E6

Forward:

(SEQ ID NO. 147)

5′-TAYSAMSTGGACCTGCAGGACC-3′

Reverse:

(SEQ ID NO. 148)

3′-GGCCWYGCATGRTKTCCAACACT-5′

E7

Forward:

(SEQ ID NO. 149)

5′-CAATTWGCCAGCTCAGAMGAGGA-3′

Reverse:

(SEQ ID NO. 150)

3′-CCACCACMAGCCTWACTGYACRV-5′

L1

Forward:

(SEQ ID NO. 151)

5′-ARGTATACCTGCCTCCYGCCC-3′

Reverse:

(SEQ ID NO. 152)

3′-CCTGTGCWCGTTGYAGCCAG-5′

As used herein, G is used to designate Guanine, A is used to designate Adenine, T is used to designate a Thymine, C is used to designate a Cytosine. R is commonly used to designate a Purine (A or G), Y is commonly used to designate a Pyrimidine (T or C), W is commonly used to designate A or T, S is commonly used to designate C or G, K is commonly used to designate G or T, H is commonly used to designate A or T or C, B is commonly used to designate G or C or T, V is commonly used to designate G or A or T, D is commonly used to designate G or A or T, N is commonly used to designate any nucleotide (A or T or C or G).

Addition of indices and sequencing adapters are needed for sequencing technologies and can be added by standard procedures. For example, said primers can be used in solution or linked to a solid support. To permit its covalent coupling to the support, the primer is generally functionalized. Thus, it may be modified by a thiol, amine or carboxyl terminal group at the 5′ or 3′ position. In particular, the addition of a thiol, amine or carboxyl group makes it possible, for example, to couple the oligonucleotide to a support bearing disulphide, maleimide, amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde functions. These couplings form by establishment of disulphide, thioether, ester, amide or amine links between the primer and the support. Any other method known to a person skilled in the art may be used, such as bifunctional coupling reagents, for example.

Moreover, to improve the hybridization with the coupled oligonucleotide, it can be advantageous for the oligonucleotide to contain an “arm” and a “spacer” sequence of bases. The use of an arm makes it possible, in effect, to bind the primer at a chosen distance from the support, enabling its conditions of interaction with the DNA to be improved. The arm advantageously consists of a linear carbon chain, comprising 1 to 18 and preferably 6 or 12 (CH₂) groups, and an amine which permits binding to the column. The arm is linked to a phosphate of the oligonucleotide or of a “spacer” composed of bases which do not interfere with the hybridization. Thus, the “spacer” can comprise purine bases. As an example, the “spacer” can comprise the sequence GAGG. The arm is advantageously composed of a linear carbon chain comprising 6 or 12 carbon atoms.

For implementation of the present invention, different types of support may be used. These can be functionalized chromatographic supports, in bulk or prepacked in a column, functionalized plastic surfaces or functionalized latex beads, magnetic or otherwise. Chromatographic supports are preferably used. As an example, the chromatographic supports capable of being used are agarose, acrylamide or dextran as well as their derivatives (such as Sephadex, Sepharose, Superose, etc.), polymers such as poly(styrene/divinylbenzene), or grafted or ungrafted silica, for example. The chromatography columns can operate in the diffusion or perfusion mode.

As used herein, the term “sequencing” is used in a broad sense and refers to any technique known by the skilled person including but not limited to Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing (MPSS), sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD® sequencing, MS-PET sequencing, mass spectrometry, and a combination thereof. In specific embodiments, the method and kit of the invention is adapted to run on ABI PRISM® 377 DNA Sequencer, an ABI PRISM® 310, 3100, 3100-Avant, 3730, or 3730x1 Genetic Analyzer, an ABI PRISM® 3700 DNA Analyzer, or an Applied Biosystems SOLiD™ System (all from Applied Biosystems), a Genome Sequencer 20 System (Roche Applied Science).

For all technologies described herein, although the said primers can be used in solution, in another embodiment the said primers are linked to a solid support.

To permit its covalent coupling to the support, the primer is generally functionalized. Thus, it may be modified by a thiol, amine or carboxyl terminal group at the 5′ or 3′ position. In particular, the addition of a thiol, amine or carboxyl group makes it possible, for example, to couple the oligonucleotide to a support bearing disulphide, maleimide, amine, carboxyl, ester, epoxide, cyanogen bromide or aldehyde functions. These couplings form by establishment of disulphide, thioether, ester, amide or amine links between the primer and the support. Any other method known to a person skilled in the art may be used, such as bifunctional coupling reagents, for example.

Moreover, to improve the hybridization with the coupled oligonucleotide, it can be advantageous for the oligonucleotide to contain an “arm” and a “spacer” sequence of bases. The use of an arm makes it possible, in effect, to bind the primer at a chosen distance from the support, enabling its conditions of interaction with the DNA to be improved. The arm advantageously consists of a linear carbon chain, comprising 1 to 18 and preferably 6 or 12 (CH2) groups, and an amine which permits binding to the column. The arm is linked to a phosphate of the oligonucleotide or of a “spacer” composed of bases which do not interfere with the hybridization. Thus, the “spacer” can comprise purine bases. As an example, the “spacer” can comprise the sequence GAGG. The arm is advantageously composed of a linear carbon chain comprising 6 or 12 carbon atoms.

As used herein, “oncogenic genes or oncogenic mRNAs” refers to genes or mRNAs which are directly or indirectly inducing cell transformation into cancer cells development. For example, oncogenic genes are used to designated E6 genes and/or E7 genes.

As used herein, “other viral mRNAs” refers to mRNAs coding for capsid proteins (L1 and L2), mRNAs coding for the growth stimulation (E5), mRNAs coding for replication/transcription (E4 and E2) and mRNAs coding for replication (E1 and E8), which are not oncogenic genes.

R ratios as used herein are defined as the relative level of an oncogenic mRNA, for example E6, E7 or oncogenic mRNAs, for example E6+E7, compared to other viral mRNAs of the same genotype or species, more particularly compared to selected mRNAs from genes coding for capsid proteins (L1 and L2), gene coding for the growth stimulation protein (E5), genes coding for replication or transcription proteins (E4, E2 and E1, E8).

For example, a reference Ratio R can be defined as

R=Σ(xE6 and/or xE7)/Σ(xL1 and/or xL2 and/or xE2 . . . )

Wherein x is a factor in the range 0-1000000,

Wherein xE6 is for example either the number of reads mapped to the gene Ex, or the number of times each nucleotide of the gene Ex is sequenced, and wherein xL1 is for example the number of reads mapped to the gene Lx, or the number of times each nucleotide of the gene Lx is sequenced.

DRAWINGS

FIG. 1 represents percentage homology between oncogenic proteins E6 HPVs HR and LR. The x-axis corresponds to HR-HPVs and y-axis represents the percentage of the nucleotide sequences homology of HPVs compared to LR HR HPVs.

FIG. 2 represents percentage homology between oncogenic proteins E7 HPVs HR and LR. The x-axis corresponds to HR-HPVs and y-axis represents the percentage of the nucleotide sequences homology of HPVs compared to LR HR HPVs.

FIG. 3 represents percentage homology between oncogenic proteins E6 HPVs HR. The x-axis corresponds to HR-HPVs and y-axis represents the percentage of the nucleotide sequences homology of HR HPVs.

FIG. 4 represents percentage homology between oncogenic proteins E7 HPVs HR. The x-axis corresponds to HR-HPVs and y-axis represents the percentage of the nucleotide sequences homology of H HPVs.

FIG. 5 represents percentage homology between oncogenic proteins E6 HPVs LR. The x-axis corresponds to HR-HPVs and y-axis represents the percentage of the nucleotide sequences homology of HR HPVs.

FIG. 6 represents percentage homology between oncogenic proteins E7 HPVs LR. The x-axis corresponds to HR-HPVs and y-axis represents the percentage of the nucleotide sequences homology of HR HPVs.

FIG. 7 represents transcription map of HR αHPV. Upper part: genomic coordinates of splice donor (SD) and acceptor (SA) sites are indicated for each HR αHPV (light grey for previously documented sites, dark grey for sites identified by analogy). Additional polyA (pA) and putative breakpoint sites (put bkpt) are added. Lower part: overview of HR αHPV splice events (black line: sequences found in mRNA; dot line: splice events) delineating splice isoforms that compose the αHPV transcripts database.

FIG. 8 presents Table 9

DETAILED DESCRIPTION

Referring to both first and second aspect, and in a first specific embodiment, the method is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58 and papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82.

In a second specific embodiment the method is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82 and papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44.

In a third specific embodiment, the above defined invention is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82, papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44 and papillomaviruses α8 comprising HPV 91, HPV 43, HPV 7, HPV 40.

In a forth specific embodiment, the above defined invention is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82, papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44, papillomaviruses α8 comprising HPV 91, HPV 43, HPV 7, HPV 40 and papillomaviruses α1 comprising HPV 42, HPV 32.

In a fifth specific embodiment, the above defined invention is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82, papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44, papillomaviruses α8 comprising HPV 91, HPV 43, HPV 7, HPV 40 papillomaviruses α1 comprising HPV 42, HPV 32 and papillomavirus α3 comprising HPV 114, HPV 84, HPV 86, HPV87, HPV 102, HPV83, HPV89, HPV 61, HPV 72, HPV 62.

In this regard, the invention also contemplates a composition of primers comprising for E6: α5: both SEQ ID NO. 44 and SEQ ID NO. 45, and all three SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 48 and; α6: SEQ ID NO. 58 or both SEQ ID NO. 59 and SEQ ID NO. 60, and both SEQ ID NO. 61 and SEQ ID NO. 62 and; α7: all three SEQ ID NO. 73, SEQ ID NO. 75 and SEQ ID NO. 76 or all three EQ ID NO. 74, SEQ ID NO. 75 and SEQ ID NO. 76, and all five SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80 and SEQ ID NO. 81 and; α10: all three SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117 and all four SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120, SEQ ID NO. 121; and α9: both SEQ ID NO. 100 and SEQ ID NO. 101 and all three SEQ ID NO. 102, SEQ ID NO. 103 and SEQ ID NO. 104; and α8: SEQ ID NO. 91, and both SEQ ID NO. 92 and SEQ ID NO. 93; and α1: SEQ ID NO. 1 and SEQ ID NO. 2; and α3: all three SEQ ID NO. 21, SEQ ID NO. 22 and SEQ ID NO. 23, and all four SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27

And comprising for L1 α5: SEQ ID NO. 53 or both SEQ ID NO. 54 and SEQ ID NO. 55, and both SEQ ID NO. 56 and SEQ ID NO. 57; and α6: SEQ ID NO. 71 and SEQ ID NO. 72; and α7: both SEQ ID NO. 87 and SEQ ID NO. 88, and both SEQ ID NO. 89 and SEQ ID NO. 90; and α10: both SEQ ID NO. 128, SEQ ID NO. 129 and SEQ ID NO. 130 or all four SEQ ID NO. 131, SEQ ID NO. 132, SEQ ID NO. 133, SEQ ID NO. 134; and α9: both SEQ ID NO. 111 and SEQ ID NO. 112, and both SEQ ID NO. 113 and SEQ ID NO. 114; and α8: SEQ ID NO. 97, and both SEQ ID NO. 98 and SEQ ID NO. 99; and α1: SEQ ID NO. 5 and SEQ ID NO. 6; and α3 SEQ ID NO. 33 and both SEQ ID NO. 34 and SEQ ID NO. 35.

Or, in this regard, the invention also contemplates a composition of primers comprising for E7: α5: both SEQ ID NO. 49, SEQ ID NO. 50 and both SEQ ID NO. 51, SEQ ID NO. 52; and α6: SEQ ID NO. 63 or SEQ ID NO. 64 or SEQ ID NO. 65 or SEQ ID NO. 66 or both SEQ ID NO. 67 and SEQ ID NO. 68, and both SEQ ID NO. 69 and SEQ ID NO. 70; and α7: SEQ ID NO. 82 or both SEQ ID NO. 83, SEQ ID NO. 84, and both SEQ ID NO. 85, SEQ ID NO. 86; and α10: all three SEQ ID NO. 122, SEQ ID NO. 123 and SEQ ID NO. 124, and all three SEQ ID NO. 125, SEQ ID NO. 126 and SEQ ID NO. 127; and α9: all three SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107, and all three SEQ ID NO. 108, SEQ ID NO. 109 and 110; and α8: SEQ ID NO. 94, and both SEQ ID NO. 95 and SEQ ID NO. 96; and α1: SEQ ID NO. 3 and SEQ ID NO. 4; and α3: both SEQ ID NO. 28 and SEQ ID NO. 29, and all three SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32,

In a sixth specific embodiment, the above defined invention is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82, papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44, papillomaviruses α8 comprising HPV 91, HPV 43, HPV 7, HPV 40 papillomaviruses α1 comprising HPV 42, HPV 32, papillomavirus α3 comprising HPV 114, HPV 84, HPV 86, HPV87, HPV 102, HPV83, HPV89, HPV 61, HPV 72, HPV 62 and papillomavirus α2 comprising HPV 117, HPV 10, HPV 94, HPV 28, HPV125, HPV 3, HPV 78, HPV 160, HPV 29, HPV 77.

Or, in this regard, the invention also contemplates a composition of primers comprising for E7: α5: both SEQ ID NO. 49, SEQ ID NO. 50 and both SEQ ID NO. 51, SEQ ID NO. 52; and α6: SEQ ID NO. 63 or SEQ ID NO. 64 or SEQ ID NO. 65 SEQ ID NO. 66 or both SEQ ID NO. 67 and SEQ ID NO. 68, and both SEQ ID NO. 69 and SEQ ID NO. 70; and α7: SEQ ID NO. 82 or both SEQ ID NO. 83, SEQ ID NO. 84, and both SEQ ID NO. 85, SEQ ID NO. 86; and α10: all three SEQ ID NO. 122, SEQ ID NO. 123 and SEQ ID NO. 124, and all three SEQ ID NO. 125, SEQ ID NO. 126 and SEQ ID NO. 127; and α9: all three SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107, and all three SEQ ID NO. 108, SEQ ID NO. 109 and 110; and α8: SEQ ID NO. 94, and both SEQ ID NO. 95 and SEQ ID NO. 96; and α1: SEQ ID NO. 3 and SEQ ID NO. 4; and α3: both SEQ ID NO. 28 and SEQ ID NO. 29, and all three SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32; and α2: all three SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and all three SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17,

In a seventh specific embodiment, the above defined invention is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82, papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44, papillomaviruses α8 comprising HPV 91, HPV 43, HPV 7, HPV 40 papillomaviruses α1 comprising HPV 42, HPV 32, papillomavirus α3 comprising HPV 114, HPV 84, HPV 86, HPV87, HPV 102, HPV83, HPV89, HPV 61, HPV 72, HPV 62, papillomavirus α2 comprising HPV 117, HPV 10, HPV 94, HPV 28, HPV125, HPV 3, HPV 78, HPV 160, HPV 29, HPV 77 and papillomaviruses α4 comprising HPV 2, HPV 27, HPV 57.

Or, in this regard, the invention also contemplates a composition of primers comprising for E7: α5: both SEQ ID NO. 49, SEQ ID NO. 50 and both SEQ ID NO. 51, SEQ ID NO. 52; and α6: SEQ ID NO. 63 or SEQ ID NO. 64 or SEQ ID NO. 65 SEQ ID NO. 66 or both SEQ ID NO. 67 and SEQ ID NO. 68, and both SEQ ID NO. 69 and SEQ ID NO. 70; and α7: SEQ ID NO. 82 or both SEQ ID NO. 83, SEQ ID NO. 84, and both SEQ ID NO. 85, SEQ ID NO. 86; and α10: all three SEQ ID NO. 122, SEQ ID NO. 123 and SEQ ID NO. 124, and all three SEQ ID NO. 125, SEQ ID NO. 126 and SEQ ID NO. 127; and α9: all three SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107, and all three SEQ ID NO. 108, SEQ ID NO. 109 and 110; and α8: SEQ ID NO. 94, and both SEQ ID NO. 95 and SEQ ID NO. 96; and α1: SEQ ID NO. 3 and SEQ ID NO. 4; and α3: both SEQ ID NO. 28 and SEQ ID NO. 29, and all three SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32; and α2: all three SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and all three SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17; and α4: SEQ ID NO. 38, and SEQ ID NO. 39

In an eighth specific embodiment, the above defined invention is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82, papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44, papillomaviruses α8 comprising HPV 91, HPV 43, HPV 7, HPV 40 papillomaviruses α1 comprising HPV 42, HPV 32, papillomavirus α3 comprising HPV 114, HPV 84, HPV 86, HPV87, HPV 102, HPV83, HPV89, HPV 61, HPV 72, HPV 62, papillomavirus α2 comprising HPV 117, HPV 10, HPV 94, HPV 28, HPV125, HPV 3, HPV 78, HPV 160, HPV 29, HPV 77, papillomaviruses α4 comprising HPV 2, HPV 27, HPV 57 and papillomaviruses all comprising HPV 73, HPV 34.

Or, in this regard, the invention also contemplates a composition of primers comprising for E7: α5: both SEQ ID NO. 49, SEQ ID NO. 50 and both SEQ ID NO. 51, SEQ ID NO. 52; and α6: SEQ ID NO. 63 or SEQ ID NO. 64 or SEQ ID NO. 65 SEQ ID NO. 66 or both SEQ ID NO. 67 and SEQ ID NO. 68, and both SEQ ID NO. 69 and SEQ ID NO. 70; and α7: SEQ ID NO. 82 or both SEQ ID NO. 83, SEQ ID NO. 84, and both SEQ ID NO. 85, SEQ ID NO. 86; and α10: all three SEQ ID NO. 122, SEQ ID NO. 123 and SEQ ID NO. 124, and all three SEQ ID NO. 125, SEQ ID NO. 126 and SEQ ID NO. 127; and α9: all three SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107, and all three SEQ ID NO. 108, SEQ ID NO. 109 and 110; and α8: SEQ ID NO. 94, and both SEQ ID NO. 95 and SEQ ID NO. 96; and α1: SEQ ID NO. 3 and SEQ ID NO. 4; and α3: both SEQ ID NO. 28 and SEQ ID NO. 29, and all three SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32; and α2: all three SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and all three SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17; and α4: SEQ ID NO. 38, and SEQ ID NO. 39; and all SEQ ID NO. 137, and SEQ ID NO. 138,

In a ninth specific embodiment, the above defined invention is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82, papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44, papillomaviruses α8 comprising HPV 91, HPV 43, HPV 7, HPV 40, papillomaviruses α1 comprising HPV 42, HPV 32, papillomavirus α3 comprising HPV 114, HPV 84, HPV 86, HPV87, HPV 102, HPV83, HPV89, HPV 61, HPV 72, HPV 62, papillomavirus α2 comprising HPV 117, HPV 10, HPV 94, HPV 28, HPV125, HPV 3, HPV 78, HPV 160, HPV 29, HPV 77, papillomaviruses α4 comprising HPV 2, HPV 27, HPV 57, papillomaviruses all comprising HPV 73, HPV 34 and papillomaviruses α13 comprising HPV 54.

Or, in this regard, the invention also contemplates a composition of primers comprising for E7: α5: both SEQ ID NO. 49, SEQ ID NO. 50 and both SEQ ID NO. 51, SEQ ID NO. 52; and α6: SEQ ID NO. 63 or SEQ ID NO. 64 or SEQ ID NO. 65 SEQ ID NO. 66 or both SEQ ID NO. 67 and SEQ ID NO. 68, and both SEQ ID NO. 69 and SEQ ID NO. 70; and α7: SEQ ID NO. 82 or both SEQ ID NO. 83, SEQ ID NO. 84, and both SEQ ID NO. 85, SEQ ID NO. 86; and α10: all three SEQ ID NO. 122, SEQ ID NO. 123 and SEQ ID NO. 124, and all three SEQ ID NO. 125, SEQ ID NO. 126 and SEQ ID NO. 127; and α9: all three SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107, and all three SEQ ID NO. 108, SEQ ID NO. 109 and 110; and α8: SEQ ID NO. 94, and both SEQ ID NO. 95 and SEQ ID NO. 96; and α1: SEQ ID NO. 3 and SEQ ID NO. 4; and α3: both SEQ ID NO. 28 and SEQ ID NO. 29, and all three SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32; and α2: all three SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and all three SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17; and α4: SEQ ID NO. 38, and SEQ ID NO. 39; and all SEQ ID NO. 137, and SEQ ID NO. 138; and α13: SEQ ID NO. 143, and SEQ ID NO. 144,

In a tenth specific embodiment, the above defined invention is practiced to include at least the group consisting of papillomaviruses α6 comprising HPV 30, HPV 53, HPV 56 and HPV 66, papillomaviruses α7 comprising HPV 68, HPV 39, HPV 70, HPV 85, HPV 59, HPV 45, HPV 18, HPV 97, papillomaviruses α10 comprising HPV 16, HPV 35, HPV 31, HPV 52, HPV 67, HPV 33, HPV 58, papillomaviruses α5 comprising HPV 26, HPV, 69, HPV 51, HPV 82, papillomaviruses α9 comprising HPV 6, HPV 11, HPV 13, HPV 1, HPV 74, HPV 44, papillomaviruses α8 comprising HPV 91, HPV 43, HPV 7, HPV 40, papillomaviruses α1 comprising HPV 42, HPV 32, papillomavirus α3 comprising HPV 114, HPV 84, HPV 86, HPV87, HPV 102, HPV83, HPV89, HPV 61, HPV 72, HPV 62, papillomavirus α2 comprising HPV 117, HPV 10, HPV 94, HPV 28, HPV125, HPV 3, HPV 78, HPV 160, HPV 29, HPV 77, papillomaviruses α4 comprising HPV 2, HPV 27, HPV 57, papillomaviruses all comprising HPV 73, HPV 34, papillomaviruses α13 comprising HPV 54 and papillomaviruses α14 comprising HPV 106, HPV 90, HPV 71.

And comprising for L1, α5: SEQ ID NO. 53 or both SEQ ID NO. 54 and SEQ ID NO. 55, and both SEQ ID NO. 56 and SEQ ID NO. 57; and α6: SEQ ID NO. 71 and SEQ ID NO. 72; and α7: both SEQ ID NO. 87 and SEQ ID NO. 88, and both SEQ ID NO. 89 and SEQ ID NO. 90; and α10: both SEQ ID NO. 128, SEQ ID NO. 129 and SEQ ID NO. 130 or all four SEQ ID NO. 131, SEQ ID NO. 132, SEQ ID NO. 133, SEQ ID NO. 134; and α9: both SEQ ID NO. 111 and SEQ ID NO. 112, and both SEQ ID NO. 113 and SEQ ID NO. 114; and α8: SEQ ID NO. 97, and both SEQ ID NO. 98 and SEQ ID NO. 99; and α1: SEQ ID NO. 5 and SEQ ID NO. 6; and α3 SEQ ID NO. 33 and both SEQ ID NO. 34 and SEQ ID NO. 35; and α2 SEQ ID NO. 18, and both SEQ ID NO. 19 and SEQ ID NO. 20; and α4: SEQ ID NO. 40 or SEQ ID NO. 41, and SEQ ID NO. 42 or SEQ ID NO. 43; and all: SEQ ID NO. 139, and SEQ ID NO. 140; and α13 SEQ ID NO. 145, and SEQ ID NO. 146; and α14: SEQ ID NO. 151 and SEQ ID NO. 152.

Or, in this regard, the invention also contemplates a composition of primers comprising for E7: α5: both SEQ ID NO. 49, SEQ ID NO. 50 and both SEQ ID NO. 51, SEQ ID NO. 52; and α6: SEQ ID NO. 63 or SEQ ID NO. 64 or SEQ ID NO. 65 SEQ ID NO. 66 or both SEQ ID NO. 67 and SEQ ID NO. 68, and both SEQ ID NO. 69 and SEQ ID NO. 70; and α7: SEQ ID NO. 82 or both SEQ ID NO. 83, SEQ ID NO. 84, and both SEQ ID NO. 85, SEQ ID NO. 86; and α10: all three SEQ ID NO. 122, SEQ ID NO. 123 and SEQ ID NO. 124, and all three SEQ ID NO. 125, SEQ ID NO. 126 and SEQ ID NO. 127; and α9: all three SEQ ID NO. 105, SEQ ID NO. 106 and SEQ ID NO. 107, and all three SEQ ID NO. 108, SEQ ID NO. 109 and 110; and α8: SEQ ID NO. 94, and both SEQ ID NO. 95 and SEQ ID NO. 96; and α1: SEQ ID NO. 3 and SEQ ID NO. 4; and α3: both SEQ ID NO. 28 and SEQ ID NO. 29, and all three SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32; and α2: all three SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, and all three SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17; and α4: SEQ ID NO. 38, and SEQ ID NO. 39; and all SEQ ID NO. 137, and SEQ ID NO. 138; and α13: SEQ ID NO. 143, and SEQ ID NO. 144; and α14: SEQ ID NO. 149, and SEQ ID NO. 150

In another embodiment, the present invention relates to the use of composition of primers describe above for diagnosis or prognosis of risk to develop HPV induced cancer in a human subject.

Said primers can further comprise at least one of:

- a functional group for covalent coupling at the 5′ or 3′ end, such as a terminal group comprising a thiol, amine or carboxyl group,
- a spacer molecule or sequence at the 5′ or 3′ end,
- additional sequences as index or tag sequences to perform pre or post additional and general amplification steps not depending on the target sequences to be quantified.

In another embodiment, the present invention relates to a composition of primers comprising at least one primer selected from SEQ ID No 153 to 158. Said composition can comprise 1, 2, 3, 4, 5 or the 6 primers selected from SEQ ID No 153 to 158.

In another embodiment, the present invention relates to a kit for diagnosis or prognosis risk to develop HPV induced cancer comprising:

a) a composition of primers,
b) reagents to detect amplification products.

In a specific embodiment, the present invention relates to a kit for diagnosis or prognosis risk to develop HPV induced cancer comprising:

a) primers or probes for detecting at least a first marker selected from E6 mRNAs of group alpha HPVs, E7 mRNAs of group alpha HPVs, or both,
b) primers or probes for detecting at least a second marker selected from L1 mRNAs of group alpha HPVs, L2 mRNAs of group alpha HPVs, or both, wherein said E6, E7, L1 and L2 mRNAs have corresponding intragenetic sequences,
c) and optionally, primers or probes for detecting at least one host cellular marker indicative of neoplasia or cancer.

In various embodiments, the primers can be selected from primers comprising or consisting of the nucleic acid sequence of any of SEQ ID NOs: 1-152. Preferably the primers comprise or consist of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides of any of SEQ ID NOs:1-152. The kit can contain any of the compositions of primers described herein.

The kit can further contain at least 1, 2, 3, 4, or more controls for the determination of R. The controls can contain a known ratio of E6 and/or E7 to L1 and/or L2. Preferably, the controls contain a known ratio of E6 and E7 to L1 and L2.

In various embodiments, the kit contains at least 1 or at least 2 controls indicating a low risk or non-persistent HPV infection. In various embodiments, the kit contains at least 1 or at least 2 controls indicative of a high risk infection or associated with higher risk of developing genital neoplasia and cancer. In preferred embodiments, the kit contains at least 1 or at least 2 controls indicating a low risk or non-persistent HPV infection and at least 1 or at least 2 controls indicating a high risk infection or associated with higher risk of developing genital neoplasia and cancer.

In various embodiments, the invention encompasses a method for assessing a human papilloma virus (HPV) infected patient. In one embodiment, the method comprises generating cDNA from a patient sample comprising RNA and sequencing the cDNA to generate reads of sequence of the cDNA.

In various embodiments, the number of reads is at least 10⁶, 5×10⁶, 10⁷, 2×10⁷, or 5×10⁷reads.

In one embodiment, the cDNA is generated using random primers. In one embodiment, the cDNA is generated using HPV-specific primers. In preferred embodiments, at least one of the primers comprises or consists of the nucleic acid sequences in Table 3.

In various embodiments, the method comprises discriminating HPV sequence reads on the basis of HPV species including any of the specific species referenced herein. In various embodiments, the method comprises discriminating HPV sequence reads on the basis of HPV gene transcript, including E1, E2, E4, E5, E6, E7, E8, L1, and L2 transcripts. The transcripts can be spliced transcripts.

In various embodiments, the cDNA or the sequencing can be performed with HPV-specific or random primers, preferably HPV-specific primers.

In various embodiments, the primers comprise or consist of any of the nucleic acid sequences of SEQ ID NOs: 1-158. In various embodiments, the cDNA is generated with HPV-specific primers and the sequencing performed randomly or specifically for HPV sequences.

In various embodiments, the cDNA is generated with random primers and the sequencing performed randomly or specifically for HPV sequences.

According to a preferred embodiment, the method comprises:

a) enrichment of the viral RNAs, preferably HPV16 RNAs, in a sample,
b) random reverse transcription reaction, advantageously performed with random hexamers,
c) amplification of the cDNA produced in step a), advantageously performed by multiplex PCR with HPV-specific primers (to generate a DNA sequence library),
d) high throughput sequencing of the DNA library produced in step c) and generating reads of said cDNA,
e) determining the number of reads matching said viruses based on species discrimination and determining the most prevalent high risk species present in the sample relative to other species,
f) determining within said most prevalent high risk species the relative number of reads matching at least one oncogenic gene compared to at least one non oncogenic genes, preferably oncogenic genes compared to non oncogenic genes,
g) computing ratios within said high risk species of reads matching at least one oncogenic gene versus at least one versus corresponding at least one interspecies structural or regulatory gene, preferably oncogenic genes versus corresponding interspecies structural or regulatory genes,
h) determining risk of developing oncogenic virus induced cancer in patients in which said ratio tend towards infinity.

In advantageous embodiments, the HPV-specific primers comprise at least one of, preferably all, the following groups of pairs of primers:

- the HPV16-specific primers comprising or consisting of the primers of SEQ ID NOs: 219-258 for HPV16 genomic and unspliced transcripts, SEQ ID NOs: 259-352 for HPV16 spliced transcripts and SEQ ID NOs: 353-376 for HPV16-human fusion transcripts (including the pairs of primers of SEQ ID NO: 219-220; 221-222; 223-224; 225-226; 227-228; 229-230; 231-232; 233-234; 235-236; 237-238; 239-240; 241-242; 243-244; 245-246; 247-248; 249-250; 251-252; 253-254; 255-256; 257-258; 259-260; 261-262; 263-264; 265-266; 267-268; 269-270; 271-272; 273-274; 275-276; 277-278; 279-280; 281-282; 283-284; 285-286; 287-288; 289-290; 291-292; 293-294; 295-296; 297-298; 299-300; 301-302; 303-304; 305-306; 307-308; 309-310; 311-312; 313-314; 315-316; 317-318; 319-320; 321-322; 323-324; 325-326; 327-328; 329-330; 331-332; 333-334; 335-336; 337-338; 339-340; 341-342; 343-344; 345-346; 347-348; 349-350; 351-352; 353-354; 355-356; 357-358; 359-360; 361-362; 363-364; 365-366; 367-368; 369-370; 371-372; 373-374; 375-376) or 377-470 (including the pairs of primers of SEQ ID NO. 377-378; 379-380; 381-382; 383-384; 385-386; 387-388; 389-390; 391-392; 393-394; 395-396; 397-398; 399-400; 401-402; 403-404; 405-406; 407-408; 409-410; 411-412; 413-414; 415-416; 417-418; 419-420; 421-422; 423-424; 425-426; 427-428; 429-430; 431-432; 433-434; 435-436; 437-438; 439-440; 441-442; 443-444; 445-446; 447-448; 449-450; 451-452; 453-454; 455-456; 457-458; 459-460; 461-462; 463-464; 465-466; 467-468 and; 469-470); and/or,
- the HPV18-specific primers comprising or consisting of the primers of SEQ ID NO. 471-574 (including the pairs of primers of SEQ ID NO.: 471-472; 473-474; 475-476; 477-478; 479-480; 481-482; 483-484; 485-486; 487-488; 489-490; 491-492; 493-494; 495-496; 497-498; 499-500; 501-502; 503-504; 505-506; 507-508; 509-510; 511-512; 513-514; 515-516; 517-518; 519-520; 521-522; 523-524; 525-526; 527-528; 529-530; 531-532; 533-534; 535-536; 537-538; 539-540; 541-542; 543-544; 545-546; 547-548; 549-550; 551-552; 553-554; 555-556; 557-558; 559-560; 561-562; 563-564; 565-566; 567-568; 569-570; 571-572; 573-574); and/or,
- the HPV31-specific primers comprising or consisting of the primers of SEQ ID NO. 575-668 (including the pairs of primers of SEQ ID NO.: 575-576; 577-578; 579-580; 581-582; 583-584; 585-586; 587-588; 589-590; 591-592; 593-594; 595-596; 597-598; 599-600; 601-602; 603-604; 605-606; 607-608; 609-610; 611-612; 613-614; 615-616; 617-618; 619-620; 621-622; 623-624; 625-626; 627-628; 629-630; 631-632; 633-634; 635-636; 637-638; 639-640; 641-642; 643-644; 645-646; 647-648; 649-650; 651-652; 653-654; 655-656; 657-658; 659-660; 661-662; 663-664; 665-666; 667-668); and/or,
- the HPV33-specific primers comprising or consisting of SEQ ID NO. 669-756 (including the pairs of primers of SEQ ID NO.: 669-670; 671-672; 673-674; 675-676; 677-678; 679-680; 681-682; 683-684; 685-686; 687-688; 689-690; 691-692; 693-694; 695-696; 697-698; 699-700; 701-702; 703-704; 705-706; 707-708; 709-710; 711-712; 713-714; 715-716; 717-718; 719-720; 721-722; 723-724; 725-726; 727-728; 729-730; 731-732; 733-734; 735-736; 737-738; 739-740; 741-742; 743-744; 745-746; 747-748; 749-750; 751-752; 753-754; 755-756); and/or,
- the HPV35-specific primers comprising or consisting of the primers of SEQ ID NO. 757-848 (including the pairs of primers of SEQ ID NO.: 757-758; 759-760; 761-762; 763-764; 765-766; 767-768; 769-770; 771-772; 773-774; 775-776; 777-778; 779-780; 781-782; 783-784; 785-786; 787-788; 789-790; 791-792; 793-794; 795-796; 797-798; 799-800; 801-802; 803-804; 805-806; 807-808; 809-810; 811-812; 813-814; 815-816; 817-818; 819-820; 821-822; 823-824; 825-826; 827-828; 829-830; 831-832; 833-834; 835-836; 837-838; 839-840; 841-842; 843-844; 845-846; 847-848); and/or,
- the HPV39-specific primers comprising or consisting of the primers of SEQ ID NO. 849-928 (including the pairs of primers of SEQ ID NO.: 849-850; 851-852; 853-854; 855-856; 857-858; 859-860; 861-862; 863-864; 865-866; 867-868; 869-870; 871-872; 873-874; 875-876; 877-878; 879-880; 881-882; 883-884; 885-886; 887-888; 889-890; 891-892; 893-894; 895-896; 897-898; 899-900; 901-902; 903-904; 905-906; 907-908; 909-910; 911-912; 913-914; 915-916; 917-918; 919-920; 921-922; 923-924; 925-926; 927-928); and/or,
- the HPV45-specific primers comprising or consisting of the primers of SEQ ID NO. 929-1020 (including the pairs of primers of SEQ ID NO.: 929-930; 931-932; 933-934; 935-936; 937-938; 939-940; 941-942; 943-944; 945-946; 947-948; 949-950; 951-952; 953-954; 955-956; 957-958; 959-960; 961-962; 963-964; 965-966; 967-968; 969-970; 971-972; 973-974; 975-976; 977-978; 979-980; 981-982; 983-984; 985-986; 987-988; 989-990; 991-992; 993-994; 995-996; 997-998; 999-1000; 1001-1002; 1003-1004; 1005-1006; 1007-1008; 1009-1010; 1011-1012; 1013-1014; 1015-1016; 1017-1018; 1019-1020); and/or,
- the HPV51-specific primers comprising or consisting of the primers of SEQ ID NO. 1021-1102 (including the pairs of primers of SEQ ID NO.: 1021-1022; 1023-1024; 1025-1026; 1027-1028; 1029-1030; 1031-1032; 1033-1034; 1035-1036; 1037-1038; 1039-1040; 1041-1042; 1043-1044; 1045-1046; 1047-1048; 1049-1050; 1051-1052; 1053-1054; 1055-1056; 1057-1058; 1059-1060; 1061-1062; 1063-1064; 1065-1066; 1067-1068; 1069-1070; 1071-1072; 1073-1074; 1075-1076; 1077-1078; 1079-1080; 1081-1082; 1083-1084; 1085-1086; 1087-1088; 1089-1090; 1091-1092; 1093-1094; 1095-1096; 1097-1098; 1099-1100; 1101-1102); and/or,
- the HPV52-specific primers comprising or consisting of the primers of SEQ ID NO. 1103-1200 (including the pairs of primers of SEQ ID NO.: 1103-1104; 1105-1106; 1107-1108; 1109-1110; 1111-1112; 1113-1114; 1115-1116; 1117-1118; 1119-1120; 1121-1122; 1123-1124; 1125-1126; 1127-1128; 1129-1130; 1131-1132; 1133-1134; 1135-1136; 1137-1138; 1139-1140; 1141-1142; 1143-1144; 1145-1146; 1147-1148; 1149-1150; 1151-1152; 1153-1154; 1155-1156; 1157-1158; 1159-1160; 1161-1162; 1163-1164; 1165-1166; 1167-1168; 1169-1170; 1171-1172; 1173-1174; 1175-1176; 1177-1178; 1179-1180; 1181-1182; 1183-1184; 1185-1186; 1187-1188; 1189-1190; 1191-1192; 1193-1194; 1195-1196; 1197-1198; 1199-1200); and/or,
- the HPV56-specific primers comprising or consisting of the primers of SEQ ID NO. 1201-1296 (including the pairs of primers of SEQ ID NO.: 1201-1202; 1203-1204; 1205-1206; 1207-1208; 1209-1210; 1211-1212; 1213-1214; 1215-1216; 1217-1218; 1219-1220; 1221-1222; 1223-1224; 1225-1226; 1227-1228; 1229-1230; 1231-1232; 1233-1234; 1235-1236; 1237-1238; 1239-1240; 1241-1242; 1243-1244; 1245-1246; 1247-1248; 1249-1250; 1251-1252; 1253-1254; 1255-1256; 1257-1258; 1259-1260; 1261-1262; 1263-1264; 1265-1266; 1267-1268; 1269-1270; 1271-1272; 1273-1274; 1275-1276; 1277-1278; 1279-1280; 1281-1282; 1283-1284; 1285-1286; 1287-1288; 1289-1290; 1291-1292; 1293-1294; 1295-1296); and/or,
- the HPV58-specific primers comprising or consisting of the primers of SEQ ID NO. 1297-1382 (including the pairs of primers of SEQ ID NO.: 1297-1298; 1299-1300; 1301-1302; 1303-1304; 1305-1306; 1307-1308; 1309-1310; 1311-1312; 1313-1314; 1315-1316; 1317-1318; 1319-1320; 1321-1322; 1323-1324; 1325-1326; 1327-1328; 1329-1330; 1331-1332; 1333-1334; 1335-1336; 1337-1338; 1339-1340; 1341-1342; 1343-1344; 1345-1346; 1347-1348; 1349-1350; 1351-1352; 1353-1354; 1355-1356; 1357-1358; 1359-1360; 1361-1362; 1363-1364; 1365-1366; 1367-1368; 1369-1370; 1371-1372; 1373-1374; 1375-1376; 1377-1378; 1379-1380; 1381-1382); and/or,
- the HPV59-specific primers comprising or consisting of the primers of SEQ ID NO. 1383-1470 (including the pairs of primers of SEQ ID NO.: 1383-1384; 1385-1386; 1387-1388; 1389-1390; 1391-1392; 1393-1394; 1395-1396; 1397-1398; 1399-1400; 1401-1402; 1403-1404; 1405-1406; 1407-1408; 1409-1410; 1411-1412; 1413-1414; 1415-1416; 1417-1418; 1419-1420; 1421-1422; 1423-1424; 1425-1426; 1427-1428; 1429-1430; 1431-1432; 1433-1434; 1435-1436; 1437-1438; 1439-1440; 1441-1442; 1443-1444; 1445-1446; 1447-1448; 1449-1450; 1451-1452; 1453-1454; 1455-1456; 1457-1458; 1459-1460; 1461-1462; 1463-1464; 1465-1466; 1467-1468; 1469-1470); and/or,
- the HPV66-specific primers comprising or consisting of the primers of SEQ ID NO. 1471-1560 (including the pairs of primers of SEQ ID NO.: 1471-1472; 1473-1474; 1475-1476; 1477-1478; 1479-1480; 1481-1482; 1483-1484; 1485-1486; 1487-1488; 1489-1490; 1491-1492; 1493-1494; 1495-1496; 1497-1498; 1499-1500; 1501-1502; 1503-1504; 1505-1506; 1507-1508; 1509-1510; 1511-1512; 1513-1514; 1515-1516; 1517-1518; 1519-1520; 1521-1522; 1523-1524; 1525-1526; 1527-1528; 1529-1530; 1531-1532; 1533-1534; 1535-1536; 1537-1538; 1539-1540; 1541-1542; 1543-1544; 1545-1546; 1547-1548; 1549-1550; 1551-1552; 1553-1554; 1555-1556; 1557-1558; 1559-1560; and/or,
- the HPV68-specific primers comprising or consisting of the primers of SEQ ID NO. 1561-1642 (including the pairs of primers of SEQ ID NO.: 1561-1562; 1563-1564; 1565-1566; 1567-1568; 1569-1570; 1571-1572; 1573-1574; 1575-1576; 1577-1578; 1579-1580; 1581-1582; 1583-1584; 1585-1586; 1587-1588; 1589-1590; 1591-1592; 1593-1594; 1595-1596; 1597-1598; 1599-1600; 1601-1602; 1603-1604; 1605-1606; 1607-1608; 1609-1610; 1611-1612; 1613-1614; 1615-1616; 1617-1618; 1619-1620; 1621-1622; 1623-1624; 1625-1626; 1627-1628; 1629-1630; 1631-1632; 1633-1634; 1635-1636; 1637-1638; 1639-1640; 1641-1642); and/or,
- the HPV73-specific primers comprising or consisting of the primers of SEQ ID NO. 1643-1732 (including the pairs of primers of SEQ ID NO.: 1643-1644; 1645-1646; 1647-1648; 1649-1650; 1651-1652; 1653-1654; 1655-1656; 1657-1658; 1659-1660; 1661-1662; 1663-1664; 1665-1666; 1667-1668; 1669-1670; 1671-1672; 1673-1674; 1675-1676; 1677-1678; 1679-1680; 1681-1682; 1683-1684; 1685-1686; 1687-1688; 1689-1690; 1691-1692; 1693-1694; 1695-1696; 1697-1698; 1699-1700; 1701-1702; 1703-1704; 1705-1706; 1707-1708; 1709-1710; 1711-1712; 1713-1714; 1715-1716; 1717-1718; 1719-1720; 1721-1722; 1723-1724; 1725-1726; 1727-1728; 1729-1730; 1731-1732); and/or,
- the HPV82-specific primers comprising or consisting of the primers of SEQ ID NO. 1733-1816 (including the pairs of primers of SEQ ID NO.: 1733-1734; 1735-1736; 1737-1738; 1739-1740; 1741-1742; 1743-1744; 1745-1746; 1747-1748; 1749-1750; 1751-1752; 1753-1754; 1755-1756; 1757-1758; 1759-1760; 1761-1762; 1763-1764; 1765-1766; 1767-1768; 1769-1770; 1771-1772; 1773-1774; 1775-1776; 1777-1778; 1779-1780; 1781-1782; 1783-1784; 1785-1786; 1787-1788; 1789-1790; 1791-1792; 1793-1794; 1795-1796; 1797-1798; 1799-1800; 1801-1802; 1803-1804; 1805-1806; 1807-1808; 1809-1810; 1811-1812; 1813-1814; 1815-1816).

In other advantageous embodiments, the HPV-specific primers comprise at least one of, preferably all, the following groups of pairs of primers:

- SD1-SA1 group consisting of the pairs of primers of SEQ ID NO: 397-398; 521-522; 609-610; 695-696; 819-820; 865-866; 947-948; 1067-1068; 1119-1120; 1267-1268; 1325-1326; 1507-1508; 1597-1598; 1655-1656; 1755-1756; and/or,
- SD1-SA2 group consisting of the pairs of primers of SEQ ID NO: 459-460; 633-634; 687-688; 1111-1112; 1235-1236; 1341-1342; 1503-1504; 1657-1658; 1797-1798; and/or,
- SD1-SA3 group consisting of the pairs of primers of SEQ ID NO: 381-382; 541-542; 599-600; 903-904; 941-942; 1047-1048; 1135-1136; 1287-1288; 1459-1460; 1473-1474; 1621-1622; 1717-1718; 1745-1746; and/or,
- SD1-SA4 group consisting of the pairs of primers of SEQ ID NO: 413-414; 551-552; 637-638; 713-714; 793-794; 857-858; 981-982; 1093-1094; 1179-1180; 1227-1228; 1319-1320; 1413-1414; 1509-1510; 1563-1564; 1709-1710; 1791-1792; and/or,
- SD1-SA5 group consisting of the pairs of primers of SEQ ID NO: 453-454; 549-550; 613-614; 747-748; 761-762; 949-950; 1163-1164; 1249-1250; 1329-1330; 1453-1454; 1501-1502; and/or,
- SD1-SA6 group consisting of the pairs of primers of SEQ ID NO: 431-432; 595-596; 719-720; 827-828; 1089-1090; 1137-1138; 1285-1286; 1353-1354; 1561-1562; 1719-1720; 1763-1764; and/or,
- SD1-SA7 group consisting of the pairs of primers of SEQ ID NO: 919-920; 1449-1450; and/or,
- SD1-SA8 group consisting of the pairs of primers of SEQ ID NO: 489-490; 963-964; 1519-1520; and/or,
- SD2-5A4 group consisting of the pairs of primers of SEQ ID NO: 387-388; 473-474; 615-616; 745-746; 815-816; 849-850; 933-934; 1091-1092; 1177-1178; 1209-1210; 1367-1368; 1437-1438; 1521-1522; 1603-1604; 1651-1652; 1779-1780; and/or,
- SD2-SA5 group consisting of the pairs of primers of SEQ ID NO: 455-456; 529-530; 629-630; 717-718; 777-778; 975-976; 1153-1154; 1273-1274; 1347-1348; 1451-1452; 1531-1532; and/or,
- SD2-SA6 group consisting of the pairs of primers of SEQ ID NO: 399-400; 645-646; 727-728; 811-812; 1079-1080; 1127-1128; 1253-1254; 1369-1370; 1615-1616; 1659-1660; 1781-1782; and/or,
- SD2-SA7 group consisting of the pairs of primers of SEQ ID NO: 531-532; 899-900; 943-944; 1411-1412; 1495-1496; and/or,
- SD2-SA9 group consisting of the pairs of primers of SEQ ID NO: 437-438; 505-506; 607-608; 739-740; 785-786; 887-888; 979-980; 1063-1064; 1185-1186; 1233-1234; 1297-1298; 1423-1424; 1491-1492; 1607-1608; 1693-1694; 1775-1776; and/or,
- SD2-SA10 group consisting of the pairs of primers of SEQ ID NO: 545-546; 831-832; 1149-1150; 1269-1270; 1427-1428; 1671-1672; and/or,
- SD3-SA4 group consisting of the pairs of primers of SEQ ID NO: 379-380; 483-484; 611-612; 721-722; 833-834; 911-912; 937-938; 1053-1054; 1139-1140; 1251-1252; 1335-1336; 1435-1436; 1487-1488; 1591-1592; 1715-1716; 1785-1786; and/or,
- SD3-SA5 group consisting of the pairs of primers of SEQ ID NO: 415-416; 493-494; 593-594; 733-734; 817-818; 993-994; 1145-1146; 1243-1244; 1337-1338; 1401-1402; 1483-1484; and/or,
- SD3-SA6 group consisting of the pairs of primers of SEQ ID NO: 435-436; 655-656; 673-674; 813-814; 1045-1046; 1173-1174; 1241-1242; 1303-1304; 1557-1558; 1627-1628; 1647-1648; 1773-1774; and/or,
- SD3-SA7 group consisting of the pairs of primers of SEQ ID NO: 855-856; 1387-1388; and/or,
- SD3-SA8 group consisting of the pairs of primers of SEQ ID NO: 511-512; 957-958; 1529-1530; and/or,
- SD5-SA9 group consisting of the pairs of primers of SEQ ID NO: 419-420; 527-528; 567-568; 587-588; 683-684; 775-776; 891-892; 999-1000; 1041-1042; 1113-1114; 1247-1248; 1371-1372; 1403-1404; 1511-1512; 1617-1618; 1677-1678; 1733-1734; and/or,
- SD5-SA10 group consisting of the pairs of primers of SEQ ID NO: 495-496; 837-838; 1183-1184; 1279-1280; 1433-1434; 1723-1724.

In other embodiments, the HPV-specific primers comprise one of the following groups of pairs of primers:

- the group of pairs of primers of SEQ ID NO: 397-398; 521-522; 609-610; 695-696; 819-820; 865-866; 947-948; 1067-1068; 1119-1120; 1267-1268; 1325-1326; 1507-1508; 1597-1598; 1655-1656; 1755-1756; 459-460; 633-634; 687-688; 1111-1112; 1235-1236; 1341-1342; 1503-1504; 1657-1658; 1797-1798; 381-382; 541-542; 599-600; 903-904; 941-942; 1047-1048; 1135-1136; 1287-1288; 1459-1460; 1473-1474; 1621-1622; 1717-1718; 1745-1746; 413-414; 551-552; 637-638; 713-714; 793-794; 857-858; 981-982; 1093-1094; 1179-1180; 1227-1228; 1319-1320; 1413-1414; 1509-1510; 1563-1564; 1709-1710; 1791-1792; 453-454; 549-550; 613-614; 747-748; 761-762; 949-950; 1163-1164; 1249-1250; 1329-1330; 1453-1454; 1501-1502; 431-432; 595-596; 719-720; 827-828; 1089-1090; 1137-1138; 1285-1286; 1353-1354; 1561-1562; 1719-1720; 1763-1764; 919-920; 1449-1450; 489-490; 963-964; 1519-1520; 387-388; 473-474; 615-616; 745-746; 815-816; 849-850; 933-934; 1091-1092; 1177-1178; 1209-1210; 1367-1368; 1437-1438; 1521-1522; 1603-1604; 1651-1652; 1779-1780; 455-456; 529-530; 629-630; 717-718; 777-778; 975-976; 1153-1154; 1273-1274; 1347-1348; 1451-1452; 1531-1532; 399-400; 645-646; 727-728; 811-812; 1079-1080; 1127-1128; 1253-1254; 1369-1370; 1615-1616; 1659-1660; 1781-1782; 531-532; 899-900; 943-944; 1411-1412; 1495-1496; 437-438; 505-506; 607-608; 739-740; 785-786; 887-888; 979-980; 1063-1064; 1185-1186; 1233-1234; 1297-1298; 1423-1424; 1491-1492; 1607-1608; 1693-1694; 1775-1776; 545-546; 831-832; 1149-1150; 1269-1270; 1427-1428; 1671-1672; 379-380; 483-484; 611-612; 721-722; 833-834; 911-912; 937-938; 1053-1054; 1139-1140; 1251-1252; 1335-1336; 1435-1436; 1487-1488; 1591-1592; 1715-1716; 1785-1786; 415-416; 493-494; 593-594; 733-734; 817-818; 993-994; 1145-1146; 1243-1244; 1337-1338; 1401-1402; 1483-1484; 435-436; 655-656; 673-674; 813-814; 1045-1046; 1173-1174; 1241-1242; 1303-1304; 1557-1558; 1627-1628; 1647-1648; 1773-1774; 855-856; 1387-1388; 511-512; 957-958; 1529-1530; 477-478; 419-420; 527-528; 567-568; 587-588; 683-684; 775-776; 891-892; 999-1000; 1041-1042; 1113-1114; 1247-1248; 1371-1372; 1403-1404; 1511-1512; 1617-1618; 1677-1678; 1733-1734; 495-496; 837-838; 1183-1184; 1279-1280; 1433-1434; 1723-1724; 1011-1012; 557-558; or,
- the group of pairs of primers of SEQ ID NO: 229-230; 233-234; 235-236; 245-246; 247-248; 249-250; 251-252; 255-256; 257-258; 265-266; 273-274; 275-276; 277-278; 279-280; 281-282; 289-290; 291-292; 295-296; 297-298; 299-300; 301-302; 303-304; 305-306; 307-308; 309-310; 311-312; 319-320; 321-322; 323-324; 325-326; 327-328; 329-330; 331-332; 333-334; 335-336; 337-338; 341-342; 343-344; 345-346; 347-348; 349-350; 351-352; 377-378; 379-380; 381-382; 383-384; 385-386; 387-388; 389-390; 391-392; 393-394; 395-396; 397-398; 399-400; 401-402; 403-404; 405-406; 407-408; 409-410; 411-412; 413-414; 415-416; 417-418; 419-420; 421-422; 423-424; 425-426; 427-428; 429-430; 431-432; 433-434; 435-436; 437-438; 439-440; 441-442; 443-444; 445-446; 447-448; 449-450; 451-452; 453-454; 455-456; 457-458; 459-460; 461-462; 463-464; 465-466; 467-468; 469-470; 471-472; 473-474; 475-476; 477-478; 479-480; 481-482; 483-484; 485-486; 487-488; 489-490; 491-492; 493-494; 495-496; 497-498; 499-500; 501-502; 503-504; 505-506; 507-508; 509-510; 511-512; 513-514; 515-516; 517-518; 519-520; 521-522; 523-524; 525-526; 527-528; 529-530; 531-532; 533-534; 535-536; 537-538; 539-540; 541-542; 543-544; 545-546; 547-548; 549-550; 551-552; 553-554; 555-556; 557-558; 559-560; 561-562; 563-564; 565-566; 567-568; 569-570; 571-572; 573-574; 575-576; 577-578; 579-580; 581-582; 583-584; 585-586; 587-588; 589-590; 591-592; 593-594; 595-596; 597-598; 599-600; 601-602; 603-604; 605-606; 607-608; 609-610; 611-612; 613-614; 615-616; 617-618; 619-620; 621-622; 623-624; 625-626; 627-628; 629-630; 631-632; 633-634; 635-636; 637-638; 639-640; 641-642; 643-644; 645-646; 647-648; 649-650; 651-652; 653-654; 655-656; 657-658; 659-660; 661-662; 663-664; 665-666; 667-668; 669-670; 671-672; 673-674; 675-676; 677-678; 679-680; 681-682; 683-684; 685-686; 687-688; 689-690; 691-692; 693-694; 695-696; 697-698; 699-700; 701-702; 703-704; 705-706; 707-708; 709-710; 711-712; 713-714; 715-716; 717-718; 719-720; 721-722; 723-724; 725-726; 727-728; 729-730; 731-732; 733-734; 735-736; 737-738; 739-740; 741-742; 743-744; 745-746; 747-748; 749-750; 751-752; 753-754; 755-756; 757-758; 759-760; 761-762; 763-764; 765-766; 767-768; 769-770; 771-772; 773-774; 775-776; 777-778; 779-780; 781-782; 783-784; 785-786; 787-788; 789-790; 791-792; 793-794; 795-796; 797-798; 799-800; 801-802; 803-804; 805-806; 807-808; 809-810; 811-812; 813-814; 815-816; 817-818; 819-820; 821-822; 823-824; 825-826; 827-828; 829-830; 831-832; 833-834; 835-836; 837-838; 839-840; 841-842; 843-844; 845-846; 847-848; 849-850; 851-852; 853-854; 855-856; 857-858; 859-860; 861-862; 863-864; 865-866; 867-868; 869-870; 871-872; 873-874; 875-876; 877-878; 879-880; 881-882; 883-884; 885-886; 887-888; 889-890; 891-892; 893-894; 895-896; 897-898; 899-900; 901-902; 903-904; 905-906; 907-908; 909-910; 911-912; 913-914; 915-916; 917-918; 919-920; 921-922; 923-924; 925-926; 927-928; 929-930; 931-932; 933-934; 935-936; 937-938; 939-940; 941-942; 943-944; 945-946; 947-948; 949-950; 951-952; 953-954; 955-956; 957-958; 959-960; 961-962; 963-964; 965-966; 967-968; 969-970; 971-972; 973-974; 975-976; 977-978; 979-980; 981-982; 983-984; 985-986; 987-988; 989-990; 991-992; 993-994; 995-996; 997-998; 999-1000; 1001-1002; 1003-1004; 1005-1006; 1007-1008; 1009-1010; 1011-1012; 1013-1014; 1015-1016; 1017-1018; 1019-1020; 1021-1022; 1023-1024; 1025-1026; 1027-1028; 1029-1030; 1031-1032; 1033-1034; 1035-1036; 1037-1038; 1039-1040; 1041-1042; 1043-1044; 1045-1046; 1047-1048; 1049-1050; 1051-1052; 1053-1054; 1055-1056; 1057-1058; 1059-1060; 1061-1062; 1063-1064; 1065-1066; 1067-1068; 1069-1070; 1071-1072; 1073-1074; 1075-1076; 1077-1078; 1079-1080; 1081-1082; 1083-1084; 1085-1086; 1087-1088; 1089-1090; 1091-1092; 1093-1094; 1095-1096; 1097-1098; 1099-1100; 1101-1102; 1103-1104; 1105-1106; 1107-1108; 1109-1110; 1111-1112; 1113-1114; 1115-1116; 1117-1118; 1119-1120; 1121-1122; 1123-1124; 1125-1126; 1127-1128; 1129-1130; 1131-1132; 1133-1134; 1135-1136; 1137-1138; 1139-1140; 1141-1142; 1143-1144; 1145-1146; 1147-1148; 1149-1150; 1151-1152; 1153-1154; 1155-1156; 1157-1158; 1159-1160; 1161-1162; 1163-1164; 1165-1166; 1167-1168; 1169-1170; 1171-1172; 1173-1174; 1175-1176; 1177-1178; 1179-1180; 1181-1182; 1183-1184; 1185-1186; 1187-1188; 1189-1190; 1191-1192; 1193-1194; 1195-1196; 1197-1198; 1199-1200; 1201-1202; 1203-1204; 1205-1206; 1207-1208; 1209-1210; 1211-1212; 1213-1214; 1215-1216; 1217-1218; 1219-1220; 1221-1222; 1223-1224; 1225-1226; 1227-1228; 1229-1230; 1231-1232; 1233-1234; 1235-1236; 1237-1238; 1239-1240; 1241-1242; 1243-1244; 1245-1246; 1247-1248; 1249-1250; 1251-1252; 1253-1254; 1255-1256; 1257-1258; 1259-1260; 1261-1262; 1263-1264; 1265-1266; 1267-1268; 1269-1270; 1271-1272; 1273-1274; 1275-1276; 1277-1278; 1279-1280; 1281-1282; 1283-1284; 1285-1286; 1287-1288; 1289-1290; 1291-1292; 1293-1294; 1295-1296; 1297-1298; 1299-1300; 1301-1302; 1303-1304; 1305-1306; 1307-1308; 1309-1310; 1311-1312; 1313-1314; 1315-1316; 1317-1318; 1319-1320; 1321-1322; 1323-1324; 1325-1326; 1327-1328; 1329-1330; 1331-1332; 1333-1334; 1335-1336; 1337-1338; 1339-1340; 1341-1342; 1343-1344; 1345-1346; 1347-1348; 1349-1350; 1351-1352; 1353-1354; 1355-1356; 1357-1358; 1359-1360; 1361-1362; 1363-1364; 1365-1366; 1367-1368; 1369-1370; 1371-1372; 1373-1374; 1375-1376; 1377-1378; 1379-1380; 1381-1382; 1383-1384; 1385-1386; 1387-1388; 1389-1390; 1391-1392; 1393-1394; 1395-1396; 1397-1398; 1399-1400; 1401-1402; 1403-1404; 1405-1406; 1407-1408; 1409-1410; 1411-1412; 1413-1414; 1415-1416; 1417-1418; 1419-1420; 1421-1422; 1423-1424; 1425-1426; 1427-1428; 1429-1430; 1431-1432; 1433-1434; 1435-1436; 1437-1438; 1439-1440; 1441-1442; 1443-1444; 1445-1446; 1447-1448; 1449-1450; 1451-1452; 1453-1454; 1455-1456; 1457-1458; 1459-1460; 1461-1462; 1463-1464; 1465-1466; 1467-1468; 1469-1470; 1471-1472; 1473-1474; 1475-1476; 1477-1478; 1479-1480; 1481-1482; 1483-1484; 1485-1486; 1487-1488; 1489-1490; 1491-1492; 1493-1494; 1495-1496; 1497-1498; 1499-1500; 1501-1502; 1503-1504; 1505-1506; 1507-1508; 1509-1510; 1511-1512; 1513-1514; 1515-1516; 1517-1518; 1519-1520; 1521-1522; 1523-1524; 1525-1526; 1527-1528; 1529-1530; 1531-1532; 1533-1534; 1535-1536; 1537-1538; 1539-1540; 1541-1542; 1543-1544; 1545-1546; 1547-1548; 1549-1550; 1551-1552; 1553-1554; 1555-1556; 1557-1558; 1559-1560; 1561-1562; 1563-1564; 1565-1566; 1567-1568; 1569-1570; 1571-1572; 1573-1574; 1575-1576; 1577-1578; 1579-1580; 1581-1582; 1583-1584; 1585-1586; 1587-1588; 1589-1590; 1591-1592; 1593-1594; 1595-1596; 1597-1598; 1599-1600; 1601-1602; 1603-1604; 1605-1606; 1607-1608; 1609-1610; 1611-1612; 1613-1614; 1615-1616; 1617-1618; 1619-1620; 1621-1622; 1623-1624; 1625-1626; 1627-1628; 1629-1630; 1631-1632; 1633-1634; 1635-1636; 1637-1638; 1639-1640; 1641-1642; 1643-1644; 1645-1646; 1647-1648; 1649-1650; 1651-1652; 1653-1654; 1655-1656; 1657-1658; 1659-1660; 1661-1662; 1663-1664; 1665-1666; 1667-1668; 1669-1670; 1671-1672; 1673-1674; 1675-1676; 1677-1678; 1679-1680; 1681-1682; 1683-1684; 1685-1686; 1687-1688; 1689-1690; 1691-1692; 1693-1694; 1695-1696; 1697-1698; 1699-1700; 1701-1702; 1703-1704; 1705-1706; 1707-1708; 1709-1710; 1711-1712; 1713-1714; 1715-1716; 1717-1718; 1719-1720; 1721-1722; 1723-1724; 1725-1726; 1727-1728; 1729-1730; 1731-1732; 1733-1734; 1735-1736; 1737-1738; 1739-1740; 1741-1742; 1743-1744; 1745-1746; 1747-1748; 1749-1750; 1751-1752; 1753-1754; 1755-1756; 1757-1758; 1759-1760; 1761-1762; 1763-1764; 1765-1766; 1767-1768; 1769-1770; 1771-1772; 1773-1774; 1775-1776; 1777-1778; 1779-1780; 1781-1782; 1783-1784; 1785-1786; 1787-1788; 1789-1790; 1791-1792; 1793-1794; 1795-1796; 1797-1798; 1799-1800; 1801-1802; 1803-1804; 1805-1806; 1807-1808; 1809-1810; 1811-1812; 1813-1814; 1815-1816.

In various embodiments, the number of HPV sequence reads according to HPV species and/or HPV gene transcript can be determined.

In one embodiment, the method comprises determining the number of HPV sequence reads of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HPV species. In one embodiment, the method comprises determining the number of HPV sequence reads of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Alpha group HPV species.

In a further embodiment, the method comprises calculating a ratio (R) of the number of reads of at least one early HPV transcript to the number of reads of at least one late HPV transcript. Preferably the HPV transcripts are Alpha group HPV species HPV transcripts, most preferably HPV16 or HPV18 transcripts.

R values can be determined using any of the various formulas presented in Table 9. In one embodiment, the ratio is calculated by calculating a ratio (R) of the number of reads of HPV E6 and/or E7 transcripts to the number of reads of HPV L1 and/or L2 transcripts. In one embodiment, the ratio is calculated by calculating a ratio (R) of the number of reads of HPV E6 and E7 transcripts to the number of reads of HPV L1 and L2 transcripts.

In some embodiments, a higher ratio (R) correlates with an increased risk of developing high-grade malignant HPV-induced cancer. In various embodiments, an R value tending towards high values above 0.5, 1, 25, 50, 100 and tending towards infinity, indicates that the viral cycle is integrated, non replicating viruses expressing high level of oncogenes E6 and E7; whereas, an R value below 0.25, 0.2, 0.1, 0.05, and tending towards 0, indicates that the viral lifecycle is replicative and expressing low levels of E6 and E7.

In some embodiments, a higher number of reads of transcripts of HPV16, HPV18, or another high-risk HPV relative to reads of transcripts of a lower risk HPV species correlates with an increased risk of developing high-grade malignant HPV-induced cancer.

In some embodiments, the method comprises determining the number of reads matching the viruses based on species discrimination and determining the most prevalent high risk species present in the sample relative to other species and/or determining within said most prevalent high risk species the relative number of reads matching oncogenic genes compared to non oncogenic genes.

In one embodiment, the invention encompasses methods for assessing a human papilloma virus (HPV) infected patient comprising generating cDNA from a patient sample comprising RNA; sequencing the cDNA; generating reads of sequence of the cDNA; discriminating HPV sequence reads on the basis of HPV gene transcript; quantitating the level of HPV sequence reads according to HPV gene transcript; determining the number of HPV sequence reads of at least one HPV early gene transcript; determining the number of HPV sequence reads of at least one HPV late gene transcript; and determining the ratio of the number of HPV sequence reads of at least one HPV early gene transcript to the number of HPV sequence reads of at least one HPV late gene transcript.

The invention also contemplates a composition of group alpha HPV-specific primers comprising at least one of, preferably all, the following groups of pairs of primers:

- the HPV16-specific primers comprising or consisting of the primers of SEQ ID NOs: 219-258 for HPV16 genomic and unspliced transcripts, SEQ ID NOs: 259-352 for HPV16 spliced transcripts and SEQ ID NOs: 353-376 for HPV16-human fusion transcripts (including the pairs of primers of SEQ ID NO: 219-220; 221-222; 223-224; 225-226; 227-228; 229-230; 231-232; 233-234; 235-236; 237-238; 239-240; 241-242; 243-244; 245-246; 247-248; 249-250; 251-252; 253-254; 255-256; 257-258; 259-260; 261-262; 263-264; 265-266; 267-268; 269-270; 271-272; 273-274; 275-276; 277-278; 279-280; 281-282; 283-284; 285-286; 287-288; 289-290; 291-292; 293-294; 295-296; 297-298; 299-300; 301-302; 303-304; 305-306; 307-308; 309-310; 311-312; 313-314; 315-316; 317-318; 319-320; 321-322; 323-324; 325-326; 327-328; 329-330; 331-332; 333-334; 335-336; 337-338; 339-340; 341-342; 343-344; 345-346; 347-348; 349-350; 351-352; 353-354; 355-356; 357-358; 359-360; 361-362; 363-364; 365-366; 367-368; 369-370; 371-372; 373-374; 375-376) or 377-470 (including the pairs of primers of SEQ ID NO. 377-378; 379-380; 381-382; 383-384; 385-386; 387-388; 389-390; 391-392; 393-394; 395-396; 397-398; 399-400; 401-402; 403-404; 405-406; 407-408; 409-410; 411-412; 413-414; 415-416; 417-418; 419-420; 421-422; 423-424; 425-426; 427-428; 429-430; 431-432; 433-434; 435-436; 437-438; 439-440; 441-442; 443-444; 445-446; 447-448; 449-450; 451-452; 453-454; 455-456; 457-458; 459-460; 461-462; 463-464; 465-466; 467-468 and; 469-470); and/or,
- the HPV18-specific primers comprising or consisting of the primers of SEQ ID NO. 471-574 (including the pairs of primers of SEQ ID NO.: 471-472; 473-474; 475-476; 477-478; 479-480; 481-482; 483-484; 485-486; 487-488; 489-490; 491-492; 493-494; 495-496; 497-498; 499-500; 501-502; 503-504; 505-506; 507-508; 509-510; 511-512; 513-514; 515-516; 517-518; 519-520; 521-522; 523-524; 525-526; 527-528; 529-530; 531-532; 533-534; 535-536; 537-538; 539-540; 541-542; 543-544; 545-546; 547-548; 549-550; 551-552; 553-554; 555-556; 557-558; 559-560; 561-562; 563-564; 565-566; 567-568; 569-570; 571-572; 573-574); and/or,
- the HPV31-specific primers comprising or consisting of the primers of SEQ ID NO. 575-668 (including the pairs of primers of SEQ ID NO.: 575-576; 577-578; 579-580; 581-582; 583-584; 585-586; 587-588; 589-590; 591-592; 593-594; 595-596; 597-598; 599-600; 601-602; 603-604; 605-606; 607-608; 609-610; 611-612; 613-614; 615-616; 617-618; 619-620; 621-622; 623-624; 625-626; 627-628; 629-630; 631-632; 633-634; 635-636; 637-638; 639-640; 641-642; 643-644; 645-646; 647-648; 649-650; 651-652; 653-654; 655-656; 657-658; 659-660; 661-662; 663-664; 665-666; 667-668); and/or,
- the HPV33-specific primers comprising or consisting of SEQ ID NO. 669-756 (including the pairs of primers of SEQ ID NO.: 669-670; 671-672; 673-674; 675-676; 677-678; 679-680; 681-682; 683-684; 685-686; 687-688; 689-690; 691-692; 693-694; 695-696; 697-698; 699-700; 701-702; 703-704; 705-706; 707-708; 709-710; 711-712; 713-714; 715-716; 717-718; 719-720; 721-722; 723-724; 725-726; 727-728; 729-730; 731-732; 733-734; 735-736; 737-738; 739-740; 741-742; 743-744; 745-746; 747-748; 749-750; 751-752; 753-754; 755-756); and/or,
- the HPV35-specific primers comprising or consisting of the primers of SEQ ID NO. 757-848 (including the pairs of primers of SEQ ID NO.: 757-758; 759-760; 761-762; 763-764; 765-766; 767-768; 769-770; 771-772; 773-774; 775-776; 777-778; 779-780; 781-782; 783-784; 785-786; 787-788; 789-790; 791-792; 793-794; 795-796; 797-798; 799-800; 801-802; 803-804; 805-806; 807-808; 809-810; 811-812; 813-814; 815-816; 817-818; 819-820; 821-822; 823-824; 825-826; 827-828; 829-830; 831-832; 833-834; 835-836; 837-838; 839-840; 841-842; 843-844; 845-846; 847-848); and/or,
- the HPV39-specific primers comprising or consisting of the primers of SEQ ID NO. 849-928 (including the pairs of primers of SEQ ID NO.: 849-850; 851-852; 853-854; 855-856; 857-858; 859-860; 861-862; 863-864; 865-866; 867-868; 869-870; 871-872; 873-874; 875-876; 877-878; 879-880; 881-882; 883-884; 885-886; 887-888; 889-890; 891-892; 893-894; 895-896; 897-898; 899-900; 901-902; 903-904; 905-906; 907-908; 909-910; 911-912; 913-914; 915-916; 917-918; 919-920; 921-922; 923-924; 925-926; 927-928); and/or,
- the HPV45-specific primers comprising or consisting of the primers of SEQ ID NO. 929-1020 (including the pairs of primers of SEQ ID NO.: 929-930; 931-932; 933-934; 935-936; 937-938; 939-940; 941-942; 943-944; 945-946; 947-948; 949-950; 951-952; 953-954; 955-956; 957-958; 959-960; 961-962; 963-964; 965-966; 967-968; 969-970; 971-972; 973-974; 975-976; 977-978; 979-980; 981-982; 983-984; 985-986; 987-988; 989-990; 991-992; 993-994; 995-996; 997-998; 999-1000; 1001-1002; 1003-1004; 1005-1006; 1007-1008; 1009-1010; 1011-1012; 1013-1014; 1015-1016; 1017-1018; 1019-1020); and/or,
- the HPV51-specific primers comprising or consisting of the primers of SEQ ID NO. 1021-1102 (including the pairs of primers of SEQ ID NO.: 1021-1022; 1023-1024; 1025-1026; 1027-1028; 1029-1030; 1031-1032; 1033-1034; 1035-1036; 1037-1038; 1039-1040; 1041-1042; 1043-1044; 1045-1046; 1047-1048; 1049-1050; 1051-1052; 1053-1054; 1055-1056; 1057-1058; 1059-1060; 1061-1062; 1063-1064; 1065-1066; 1067-1068; 1069-1070; 1071-1072; 1073-1074; 1075-1076; 1077-1078; 1079-1080; 1081-1082; 1083-1084; 1085-1086; 1087-1088; 1089-1090; 1091-1092; 1093-1094; 1095-1096; 1097-1098; 1099-1100; 1101-1102); and/or,
- the HPV52-specific primers comprising or consisting of the primers of SEQ ID NO. 1103-1200 (including the pairs of primers of SEQ ID NO.: 1103-1104; 1105-1106; 1107-1108; 1109-1110; 1111-1112; 1113-1114; 1115-1116; 1117-1118; 1119-1120; 1121-1122; 1123-1124; 1125-1126; 1127-1128; 1129-1130; 1131-1132; 1133-1134; 1135-1136; 1137-1138; 1139-1140; 1141-1142; 1143-1144; 1145-1146; 1147-1148; 1149-1150; 1151-1152; 1153-1154; 1155-1156; 1157-1158; 1159-1160; 1161-1162; 1163-1164; 1165-1166; 1167-1168; 1169-1170; 1171-1172; 1173-1174; 1175-1176; 1177-1178; 1179-1180; 1181-1182; 1183-1184; 1185-1186; 1187-1188; 1189-1190; 1191-1192; 1193-1194; 1195-1196; 1197-1198; 1199-1200); and/or,
- the HPV56-specific primers comprising or consisting of the primers of SEQ ID NO. 1201-1296 (including the pairs of primers of SEQ ID NO.: 1201-1202; 1203-1204; 1205-1206; 1207-1208; 1209-1210; 1211-1212; 1213-1214; 1215-1216; 1217-1218; 1219-1220; 1221-1222; 1223-1224; 1225-1226; 1227-1228; 1229-1230; 1231-1232; 1233-1234; 1235-1236; 1237-1238; 1239-1240; 1241-1242; 1243-1244; 1245-1246; 1247-1248; 1249-1250; 1251-1252; 1253-1254; 1255-1256; 1257-1258; 1259-1260; 1261-1262; 1263-1264; 1265-1266; 1267-1268; 1269-1270; 1271-1272; 1273-1274; 1275-1276; 1277-1278; 1279-1280; 1281-1282; 1283-1284; 1285-1286; 1287-1288; 1289-1290; 1291-1292; 1293-1294; 1295-1296); and/or,
- the HPV58-specific primers comprising or consisting of the primers of SEQ ID NO. 1297-1382 (including the pairs of primers of SEQ ID NO.: 1297-1298; 1299-1300; 1301-1302; 1303-1304; 1305-1306; 1307-1308; 1309-1310; 1311-1312; 1313-1314; 1315-1316; 1317-1318; 1319-1320; 1321-1322; 1323-1324; 1325-1326; 1327-1328; 1329-1330; 1331-1332; 1333-1334; 1335-1336; 1337-1338; 1339-1340; 1341-1342; 1343-1344; 1345-1346; 1347-1348; 1349-1350; 1351-1352; 1353-1354; 1355-1356; 1357-1358; 1359-1360; 1361-1362; 1363-1364; 1365-1366; 1367-1368; 1369-1370; 1371-1372; 1373-1374; 1375-1376; 1377-1378; 1379-1380; 1381-1382); and/or,
- the HPV59-specific primers comprising or consisting of the primers of SEQ ID NO. 1383-1470 (including the pairs of primers of SEQ ID NO.: 1383-1384; 1385-1386; 1387-1388; 1389-1390; 1391-1392; 1393-1394; 1395-1396; 1397-1398; 1399-1400; 1401-1402; 1403-1404; 1405-1406; 1407-1408; 1409-1410; 1411-1412; 1413-1414; 1415-1416; 1417-1418; 1419-1420; 1421-1422; 1423-1424; 1425-1426; 1427-1428; 1429-1430; 1431-1432; 1433-1434; 1435-1436; 1437-1438; 1439-1440; 1441-1442; 1443-1444; 1445-1446; 1447-1448; 1449-1450; 1451-1452; 1453-1454; 1455-1456; 1457-1458; 1459-1460; 1461-1462; 1463-1464; 1465-1466; 1467-1468; 1469-1470); and/or,
- the HPV66-specific primers comprising or consisting of the primers of SEQ ID NO. 1471-1560 (including the pairs of primers of SEQ ID NO.: 1471-1472; 1473-1474; 1475-1476; 1477-1478; 1479-1480; 1481-1482; 1483-1484; 1485-1486; 1487-1488; 1489-1490; 1491-1492; 1493-1494; 1495-1496; 1497-1498; 1499-1500; 1501-1502; 1503-1504; 1505-1506; 1507-1508; 1509-1510; 1511-1512; 1513-1514; 1515-1516; 1517-1518; 1519-1520; 1521-1522; 1523-1524; 1525-1526; 1527-1528; 1529-1530; 1531-1532; 1533-1534; 1535-1536; 1537-1538; 1539-1540; 1541-1542; 1543-1544; 1545-1546; 1547-1548; 1549-1550; 1551-1552; 1553-1554; 1555-1556; 1557-1558; 1559-1560; and/or,
- the HPV68-specific primers comprising or consisting of the primers of SEQ ID NO. 1561-1642 (including the pairs of primers of SEQ ID NO.: 1561-1562; 1563-1564; 1565-1566; 1567-1568; 1569-1570; 1571-1572; 1573-1574; 1575-1576; 1577-1578; 1579-1580; 1581-1582; 1583-1584; 1585-1586; 1587-1588; 1589-1590; 1591-1592; 1593-1594; 1595-1596; 1597-1598; 1599-1600; 1601-1602; 1603-1604; 1605-1606; 1607-1608; 1609-1610; 1611-1612; 1613-1614; 1615-1616; 1617-1618; 1619-1620; 1621-1622; 1623-1624; 1625-1626; 1627-1628; 1629-1630; 1631-1632; 1633-1634; 1635-1636; 1637-1638; 1639-1640; 1641-1642); and/or,
- the HPV73-specific primers comprising or consisting of the primers of SEQ ID NO. 1643-1732 (including the pairs of primers of SEQ ID NO.: 1643-1644; 1645-1646; 1647-1648; 1649-1650; 1651-1652; 1653-1654; 1655-1656; 1657-1658; 1659-1660; 1661-1662; 1663-1664; 1665-1666; 1667-1668; 1669-1670; 1671-1672; 1673-1674; 1675-1676; 1677-1678; 1679-1680; 1681-1682; 1683-1684; 1685-1686; 1687-1688; 1689-1690; 1691-1692; 1693-1694; 1695-1696; 1697-1698; 1699-1700; 1701-1702; 1703-1704; 1705-1706; 1707-1708; 1709-1710; 1711-1712; 1713-1714; 1715-1716; 1717-1718; 1719-1720; 1721-1722; 1723-1724; 1725-1726; 1727-1728; 1729-1730; 1731-1732); and/or,
- the HPV82-specific primers comprising or consisting of the primers of SEQ ID NO. 1733-1816 (including the pairs of primers of SEQ ID NO.: 1733-1734; 1735-1736; 1737-1738; 1739-1740; 1741-1742; 1743-1744; 1745-1746; 1747-1748; 1749-1750; 1751-1752; 1753-1754; 1755-1756; 1757-1758; 1759-1760; 1761-1762; 1763-1764; 1765-1766; 1767-1768; 1769-1770; 1771-1772; 1773-1774; 1775-1776; 1777-1778; 1779-1780; 1781-1782; 1783-1784; 1785-1786; 1787-1788; 1789-1790; 1791-1792; 1793-1794; 1795-1796; 1797-1798; 1799-1800; 1801-1802; 1803-1804; 1805-1806; 1807-1808; 1809-1810; 1811-1812; 1813-1814; 1815-1816).

The invention also contemplates a composition of pairs of group alpha HPV-specific primers comprising at least one of, preferably all, the following groups of pairs of primers:

- SD1-SA1 group consisting of the pairs of primers of SEQ ID NO: 397-398; 521-522; 609-610; 695-696; 819-820; 865-866; 947-948; 1067-1068; 1119-1120; 1267-1268; 1325-1326; 1507-1508; 1597-1598; 1655-1656; 1755-1756; and/or,
- SD1-SA2 group consisting of the pairs of primers of SEQ ID NO: 459-460; 633-634; 687-688; 1111-1112; 1235-1236; 1341-1342; 1503-1504; 1657-1658; 1797-1798; and/or,
- SD1-SA3 group consisting of the pairs of primers of SEQ ID NO: 381-382; 541-542; 599-600; 903-904; 941-942; 1047-1048; 1135-1136; 1287-1288; 1459-1460; 1473-1474; 1621-1622; 1717-1718; 1745-1746; and/or,
- SD1-SA4 group consisting of the pairs of primers of SEQ ID NO: 413-414; 551-552; 637-638; 713-714; 793-794; 857-858; 981-982; 1093-1094; 1179-1180; 1227-1228; 1319-1320; 1413-1414; 1509-1510; 1563-1564; 1709-1710; 1791-1792; and/or,
- SD1-SA5 group consisting of the pairs of primers of SEQ ID NO: 453-454; 549-550; 613-614; 747-748; 761-762; 949-950; 1163-1164; 1249-1250; 1329-1330; 1453-1454; 1501-1502; and/or,
- SD1-SA6 group consisting of the pairs of primers of SEQ ID NO: 431-432; 595-596; 719-720; 827-828; 1089-1090; 1137-1138; 1285-1286; 1353-1354; 1561-1562; 1719-1720; 1763-1764; and/or,
- SD1-SA7 group consisting of the pairs of primers of SEQ ID NO: 919-920; 1449-1450; and/or,
- SD1-SA8 group consisting of the pairs of primers of SEQ ID NO: 489-490; 963-964; 1519-1520; and/or,
- SD2-SA4 group consisting of the pairs of primers of SEQ ID NO: 387-388; 473-474; 615-616; 745-746; 815-816; 849-850; 933-934; 1091-1092; 1177-1178; 1209-1210; 1367-1368; 1437-1438; 1521-1522; 1603-1604; 1651-1652; 1779-1780; and/or,
- SD2-SA5 group consisting of the pairs of primers of SEQ ID NO: 455-456; 529-530; 629-630; 717-718; 777-778; 975-976; 1153-1154; 1273-1274; 1347-1348; 1451-1452; 1531-1532; and/or,
- SD2-SA6 group consisting of the pairs of primers of SEQ ID NO: 399-400; 645-646; 727-728; 811-812; 1079-1080; 1127-1128; 1253-1254; 1369-1370; 1615-1616; 1659-1660; 1781-1782; and/or,
- SD2-SA7 group consisting of the pairs of primers of SEQ ID NO: 531-532; 899-900; 943-944; 1411-1412; 1495-1496; and/or,
- SD2-SA9 group consisting of the pairs of primers of SEQ ID NO: 437-438; 505-506; 607-608; 739-740; 785-786; 887-888; 979-980; 1063-1064; 1185-1186; 1233-1234; 1297-1298; 1423-1424; 1491-1492; 1607-1608; 1693-1694; 1775-1776; and/or,
- SD2-SA10 group consisting of the pairs of primers of SEQ ID NO: 545-546; 831-832; 1149-1150; 1269-1270; 1427-1428; 1671-1672; and/or,
- SD3-5A4 group consisting of the pairs of primers of SEQ ID NO: 379-380; 483-484; 611-612; 721-722; 833-834; 911-912; 937-938; 1053-1054; 1139-1140; 1251-1252; 1335-1336; 1435-1436; 1487-1488; 1591-1592; 1715-1716; 1785-1786; and/or,
- SD3-SA5 group consisting of the pairs of primers of SEQ ID NO: 415-416; 493-494; 593-594; 733-734; 817-818; 993-994; 1145-1146; 1243-1244; 1337-1338; 1401-1402; 1483-1484; and/or,
- SD3-SA6 group consisting of the pairs of primers of SEQ ID NO: 435-436; 655-656; 673-674; 813-814; 1045-1046; 1173-1174; 1241-1242; 1303-1304; 1557-1558; 1627-1628; 1647-1648; 1773-1774; and/or,
- SD3-SA7 group consisting of the pairs of primers of SEQ ID NO: 855-856; 1387-1388; and/or,
- SD3-SA8 group consisting of the pairs of primers of SEQ ID NO: 511-512; 957-958; 1529-1530; and/or,
- SD5-SA9 group consisting of the pairs of primers of SEQ ID NO: 419-420; 527-528; 567-568; 587-588; 683-684; 775-776; 891-892; 999-1000; 1041-1042; 1113-1114; 1247-1248; 1371-1372; 1403-1404; 1511-1512; 1617-1618; 1677-1678; 1733-1734; and/or,
- SD5-SA10 group consisting of the pairs of primers of SEQ ID NO: 495-496; 837-838; 1183-1184; 1279-1280; 1433-1434; 1723-1724.

The invention also contemplates a composition of group alpha HPV-specific primers comprising one of the following groups of pairs of primers:

- the group of pairs of primers of SEQ ID NO: 397-398; 521-522; 609-610; 695-696; 819-820; 865-866; 947-948; 1067-1068; 1119-1120; 1267-1268; 1325-1326; 1507-1508; 1597-1598; 1655-1656; 1755-1756; 459-460; 633-634; 687-688; 1111-1112; 1235-1236; 1341-1342; 1503-1504; 1657-1658; 1797-1798; 381-382; 541-542; 599-600; 903-904; 941-942; 1047-1048; 1135-1136; 1287-1288; 1459-1460; 1473-1474; 1621-1622; 1717-1718; 1745-1746; 413-414; 551-552; 637-638; 713-714; 793-794; 857-858; 981-982; 1093-1094; 1179-1180; 1227-1228; 1319-1320; 1413-1414; 1509-1510; 1563-1564; 1709-1710; 1791-1792; 453-454; 549-550; 613-614; 747-748; 761-762; 949-950; 1163-1164; 1249-1250; 1329-1330; 1453-1454; 1501-1502; 431-432; 595-596; 719-720; 827-828; 1089-1090; 1137-1138; 1285-1286; 1353-1354; 1561-1562; 1719-1720; 1763-1764; 919-920; 1449-1450; 489-490; 963-964; 1519-1520; 387-388; 473-474; 615-616; 745-746; 815-816; 849-850; 933-934; 1091-1092; 1177-1178; 1209-1210; 1367-1368; 1437-1438; 1521-1522; 1603-1604; 1651-1652; 1779-1780; 455-456; 529-530; 629-630; 717-718; 777-778; 975-976; 1153-1154; 1273-1274; 1347-1348; 1451-1452; 1531-1532; 399-400; 645-646; 727-728; 811-812; 1079-1080; 1127-1128; 1253-1254; 1369-1370; 1615-1616; 1659-1660; 1781-1782; 531-532; 899-900; 943-944; 1411-1412; 1495-1496; 437-438; 505-506; 607-608; 739-740; 785-786; 887-888; 979-980; 1063-1064; 1185-1186; 1233-1234; 1297-1298; 1423-1424; 1491-1492; 1607-1608; 1693-1694; 1775-1776; 545-546; 831-832; 1149-1150; 1269-1270; 1427-1428; 1671-1672; 379-380; 483-484; 611-612; 721-722; 833-834; 911-912; 937-938; 1053-1054; 1139-1140; 1251-1252; 1335-1336; 1435-1436; 1487-1488; 1591-1592; 1715-1716; 1785-1786; 415-416; 493-494; 593-594; 733-734; 817-818; 993-994; 1145-1146; 1243-1244; 1337-1338; 1401-1402; 1483-1484; 435-436; 655-656; 673-674; 813-814; 1045-1046; 1173-1174; 1241-1242; 1303-1304; 1557-1558; 1627-1628; 1647-1648; 1773-1774; 855-856; 1387-1388; 511-512; 957-958; 1529-1530; 477-478; 419-420; 527-528; 567-568; 587-588; 683-684; 775-776; 891-892; 999-1000; 1041-1042; 1113-1114; 1247-1248; 1371-1372; 1403-1404; 1511-1512; 1617-1618; 1677-1678; 1733-1734; 495-496; 837-838; 1183-1184; 1279-1280; 1433-1434; 1723-1724; 1011-1012; 557-558; or,
- the group of pairs of primers of SEQ ID NO: 229-230; 233-234; 235-236; 245-246; 247-248; 249-250; 251-252; 255-256; 257-258; 265-266; 273-274; 275-276; 277-278; 279-280; 281-282; 289-290; 291-292; 295-296; 297-298; 299-300; 301-302; 303-304; 305-306; 307-308; 309-310; 311-312; 319-320; 321-322; 323-324; 325-326; 327-328; 329-330; 331-332; 333-334; 335-336; 337-338; 341-342; 343-344; 345-346; 347-348; 349-350; 351-352; 377-378; 379-380; 381-382; 383-384; 385-386; 387-388; 389-390; 391-392; 393-394; 395-396; 397-398; 399-400; 401-402; 403-404; 405-406; 407-408; 409-410; 411-412; 413-414; 415-416; 417-418; 419-420; 421-422; 423-424; 425-426; 427-428; 429-430; 431-432; 433-434; 435-436; 437-438; 439-440; 441-442; 443-444; 445-446; 447-448; 449-450; 451-452; 453-454; 455-456; 457-458; 459-460; 461-462; 463-464; 465-466; 467-468; 469-470; 471-472; 473-474; 475-476; 477-478; 479-480; 481-482; 483-484; 485-486; 487-488; 489-490; 491-492; 493-494; 495-496; 497-498; 499-500; 501-502; 503-504; 505-506; 507-508; 509-510; 511-512; 513-514; 515-516; 517-518; 519-520; 521-522; 523-524; 525-526; 527-528; 529-530; 531-532; 533-534; 535-536; 537-538; 539-540; 541-542; 543-544; 545-546; 547-548; 549-550; 551-552; 553-554; 555-556; 557-558; 559-560; 561-562; 563-564; 565-566; 567-568; 569-570; 571-572; 573-574; 575-576; 577-578; 579-580; 581-582; 583-584; 585-586; 587-588; 589-590; 591-592; 593-594; 595-596; 597-598; 599-600; 601-602; 603-604; 605-606; 607-608; 609-610; 611-612; 613-614; 615-616; 617-618; 619-620; 621-622; 623-624; 625-626; 627-628; 629-630; 631-632; 633-634; 635-636; 637-638; 639-640; 641-642; 643-644; 645-646; 647-648; 649-650; 651-652; 653-654; 655-656; 657-658; 659-660; 661-662; 663-664; 665-666; 667-668; 669-670; 671-672; 673-674; 675-676; 677-678; 679-680; 681-682; 683-684; 685-686; 687-688; 689-690; 691-692; 693-694; 695-696; 697-698; 699-700; 701-702; 703-704; 705-706; 707-708; 709-710; 711-712; 713-714; 715-716; 717-718; 719-720; 721-722; 723-724; 725-726; 727-728; 729-730; 731-732; 733-734; 735-736; 737-738; 739-740; 741-742; 743-744; 745-746; 747-748; 749-750; 751-752; 753-754; 755-756; 757-758; 759-760; 761-762; 763-764; 765-766; 767-768; 769-770; 771-772; 773-774; 775-776; 777-778; 779-780; 781-782; 783-784; 785-786; 787-788; 789-790; 791-792; 793-794; 795-796; 797-798; 799-800; 801-802; 803-804; 805-806; 807-808; 809-810; 811-812; 813-814; 815-816; 817-818; 819-820; 821-822; 823-824; 825-826; 827-828; 829-830; 831-832; 833-834; 835-836; 837-838; 839-840; 841-842; 843-844; 845-846; 847-848; 849-850; 851-852; 853-854; 855-856; 857-858; 859-860; 861-862; 863-864; 865-866; 867-868; 869-870; 871-872; 873-874; 875-876; 877-878; 879-880; 881-882; 883-884; 885-886; 887-888; 889-890; 891-892; 893-894; 895-896; 897-898; 899-900; 901-902; 903-904; 905-906; 907-908; 909-910; 911-912; 913-914; 915-916; 917-918; 919-920; 921-922; 923-924; 925-926; 927-928; 929-930; 931-932; 933-934; 935-936; 937-938; 939-940; 941-942; 943-944; 945-946; 947-948; 949-950; 951-952; 953-954; 955-956; 957-958; 959-960; 961-962; 963-964; 965-966; 967-968; 969-970; 971-972; 973-974; 975-976; 977-978; 979-980; 981-982; 983-984; 985-986; 987-988; 989-990; 991-992; 993-994; 995-996; 997-998; 999-1000; 1001-1002; 1003-1004; 1005-1006; 1007-1008; 1009-1010; 1011-1012; 1013-1014; 1015-1016; 1017-1018; 1019-1020; 1021-1022; 1023-1024; 1025-1026; 1027-1028; 1029-1030; 1031-1032; 1033-1034; 1035-1036; 1037-1038; 1039-1040; 1041-1042; 1043-1044; 1045-1046; 1047-1048; 1049-1050; 1051-1052; 1053-1054; 1055-1056; 1057-1058; 1059-1060; 1061-1062; 1063-1064; 1065-1066; 1067-1068; 1069-1070; 1071-1072; 1073-1074; 1075-1076; 1077-1078; 1079-1080; 1081-1082; 1083-1084; 1085-1086; 1087-1088; 1089-1090; 1091-1092; 1093-1094; 1095-1096; 1097-1098; 1099-1100; 1101-1102; 1103-1104; 1105-1106; 1107-1108; 1109-1110; 1111-1112; 1113-1114; 1115-1116; 1117-1118; 1119-1120; 1121-1122; 1123-1124; 1125-1126; 1127-1128; 1129-1130; 1131-1132; 1133-1134; 1135-1136; 1137-1138; 1139-1140; 1141-1142; 1143-1144; 1145-1146; 1147-1148; 1149-1150; 1151-1152; 1153-1154; 1155-1156; 1157-1158; 1159-1160; 1161-1162; 1163-1164; 1165-1166; 1167-1168; 1169-1170; 1171-1172; 1173-1174; 1175-1176; 1177-1178; 1179-1180; 1181-1182; 1183-1184; 1185-1186; 1187-1188; 1189-1190; 1191-1192; 1193-1194; 1195-1196; 1197-1198; 1199-1200; 1201-1202; 1203-1204; 1205-1206; 1207-1208; 1209-1210; 1211-1212; 1213-1214; 1215-1216; 1217-1218; 1219-1220; 1221-1222; 1223-1224; 1225-1226; 1227-1228; 1229-1230; 1231-1232; 1233-1234; 1235-1236; 1237-1238; 1239-1240; 1241-1242; 1243-1244; 1245-1246; 1247-1248; 1249-1250; 1251-1252; 1253-1254; 1255-1256; 1257-1258; 1259-1260; 1261-1262; 1263-1264; 1265-1266; 1267-1268; 1269-1270; 1271-1272; 1273-1274; 1275-1276; 1277-1278; 1279-1280; 1281-1282; 1283-1284; 1285-1286; 1287-1288; 1289-1290; 1291-1292; 1293-1294; 1295-1296; 1297-1298; 1299-1300; 1301-1302; 1303-1304; 1305-1306; 1307-1308; 1309-1310; 1311-1312; 1313-1314; 1315-1316; 1317-1318; 1319-1320; 1321-1322; 1323-1324; 1325-1326; 1327-1328; 1329-1330; 1331-1332; 1333-1334; 1335-1336; 1337-1338; 1339-1340; 1341-1342; 1343-1344; 1345-1346; 1347-1348; 1349-1350; 1351-1352; 1353-1354; 1355-1356; 1357-1358; 1359-1360; 1361-1362; 1363-1364; 1365-1366; 1367-1368; 1369-1370; 1371-1372; 1373-1374; 1375-1376; 1377-1378; 1379-1380; 1381-1382; 1383-1384; 1385-1386; 1387-1388; 1389-1390; 1391-1392; 1393-1394; 1395-1396; 1397-1398; 1399-1400; 1401-1402; 1403-1404; 1405-1406; 1407-1408; 1409-1410; 1411-1412; 1413-1414; 1415-1416; 1417-1418; 1419-1420; 1421-1422; 1423-1424; 1425-1426; 1427-1428; 1429-1430; 1431-1432; 1433-1434; 1435-1436; 1437-1438; 1439-1440; 1441-1442; 1443-1444; 1445-1446; 1447-1448; 1449-1450; 1451-1452; 1453-1454; 1455-1456; 1457-1458; 1459-1460; 1461-1462; 1463-1464; 1465-1466; 1467-1468; 1469-1470; 1471-1472; 1473-1474; 1475-1476; 1477-1478; 1479-1480; 1481-1482; 1483-1484; 1485-1486; 1487-1488; 1489-1490; 1491-1492; 1493-1494; 1495-1496; 1497-1498; 1499-1500; 1501-1502; 1503-1504; 1505-1506; 1507-1508; 1509-1510; 1511-1512; 1513-1514; 1515-1516; 1517-1518; 1519-1520; 1521-1522; 1523-1524; 1525-1526; 1527-1528; 1529-1530; 1531-1532; 1533-1534; 1535-1536; 1537-1538; 1539-1540; 1541-1542; 1543-1544; 1545-1546; 1547-1548; 1549-1550; 1551-1552; 1553-1554; 1555-1556; 1557-1558; 1559-1560; 1561-1562; 1563-1564; 1565-1566; 1567-1568; 1569-1570; 1571-1572; 1573-1574; 1575-1576; 1577-1578; 1579-1580; 1581-1582; 1583-1584; 1585-1586; 1587-1588; 1589-1590; 1591-1592; 1593-1594; 1595-1596; 1597-1598; 1599-1600; 1601-1602; 1603-1604; 1605-1606; 1607-1608; 1609-1610; 1611-1612; 1613-1614; 1615-1616; 1617-1618; 1619-1620; 1621-1622; 1623-1624; 1625-1626; 1627-1628; 1629-1630; 1631-1632; 1633-1634; 1635-1636; 1637-1638; 1639-1640; 1641-1642; 1643-1644; 1645-1646; 1647-1648; 1649-1650; 1651-1652; 1653-1654; 1655-1656; 1657-1658; 1659-1660; 1661-1662; 1663-1664; 1665-1666; 1667-1668; 1669-1670; 1671-1672; 1673-1674; 1675-1676; 1677-1678; 1679-1680; 1681-1682; 1683-1684; 1685-1686; 1687-1688; 1689-1690; 1691-1692; 1693-1694; 1695-1696; 1697-1698; 1699-1700; 1701-1702; 1703-1704; 1705-1706; 1707-1708; 1709-1710; 1711-1712; 1713-1714; 1715-1716; 1717-1718; 1719-1720; 1721-1722; 1723-1724; 1725-1726; 1727-1728; 1729-1730; 1731-1732; 1733-1734; 1735-1736; 1737-1738; 1739-1740; 1741-1742; 1743-1744; 1745-1746; 1747-1748; 1749-1750; 1751-1752; 1753-1754; 1755-1756; 1757-1758; 1759-1760; 1761-1762; 1763-1764; 1765-1766; 1767-1768; 1769-1770; 1771-1772; 1773-1774; 1775-1776; 1777-1778; 1779-1780; 1781-1782; 1783-1784; 1785-1786; 1787-1788; 1789-1790; 1791-1792; 1793-1794; 1795-1796; 1797-1798; 1799-1800; 1801-1802; 1803-1804; 1805-1806; 1807-1808; 1809-1810; 1811-1812; 1813-1814; 1815-1816.

The following examples are not limitative.

Example 1: General Amplification α5, α6, α7 and α10 HPVs

We developed a NGS test for quantifying oncogenic HPV E7 mRNAs relative their respective L1 mRNAs. We searched and designed consensus primers in this regard for a quantitative pre-amplification of the oncogenic E7 HPVs mRNAs of all alpha papillomavirus (high and low risk).

We completed the test with the identification of L1 mRNA level and determine the ratio between the expression E7 (early gene) and L1 (late gene) to determine the risk of a patient developing cancer. This new test allows determining the inherent risk of any HPVs types regardless of the current classification regardless of whether the patient is infected with HPV species 16 or 18, etc. (HR) or 30 . . . etc. (BR). Furthermore, this assay allows identifications of multiple HPV infections in single individuals.

First, we analyzed the feasibility of generating consensus sequences for different HPVs type (HR and LR).

We started to determine the percentage protein homology of the different HPVs Type, using NCBI Blastn. We observed that a high heterogeneity exists between the gene sequence of E7 from one type of HPV to another even among HR species (FIG. 1, 2, 3, 4, 5, 6). This observation did not reveal obvious features in sequences of HPVs explaining the consensus designing partners by current classification as LR or HR. this observation reinforces the hypothesis of a lack of correlation between sequence of a whole E7 gene and the classification based on the risk: this impair a possibility to derive tests based on consensus sequence to differentiate HT=R and LR.

Then, we did a sequence alignment based on oncogenic E7 HPVs HR and LR.

We focus on global comparison of HR and LR, then only within HR and finally only within LR. We observed that no specific consensus sequence emerged from the sequence alignment of HR, HR and LR/LR overall on E7 genes species. There is very little global sequence homology, the locus of homology being very punctual.

Nonetheless, we were led to design consensus primers based on subgroup a for E7 using ClustalW for alignments. We found some homologies between HPVs types sequences contained in a subgroup.

After having generated all the FASTA files alignment, we searched to generate consensus sequences using GEMI program. Sometimes, we couldn't find any consensus sequences in all the HPV subgroup.

We divided the subgroup to generate the degenerated consensus sequences. We then selected more particularly combined primers which cover all subgroup parameters such as minimizing the number of required primers selecting primers to obtain the largest possible amplicons, further selecting primers for relative conserved 3′ sequences and selecting primers which do not cross hybridize.

We did these selections for all the E7 and L1 HPVs subgroup. After analyzing all the sequences we elected the best set of primers for α5, α6, α7 and α10 E7 and α5, α6, α7 and α10 L1 to amplify all the genes.

Finally, we provide here a new diagnosis test comprising a set of probes for the pre amplification of E7 α5, α6, α7 and α10 HPVs mRNAs and L1 α5, α6, α7 and α10 HPVs mRNAs level and which allows to assess the ratio between the expression of E7 (early gene) and L1 (late gene) as a marker of the risk for a patient developing cancer.

One preferred set of primers for the pre-amplification comprises the following sequences:

α5:

E7

Forward:

(SEQ ID NO. 49)

5′-YTAGATYTGGTGCCGCAACCCG-3′

Forward:

(SEQ ID NO. 50)

5′-MGCCATGCGTGGTAATGTACCAC-3′

Reverse:

(SEQ ID NO. 51)

3′-CTCCASCRCTCGRACGTTCTGT-5′

Reverse:

(SEQ ID NO. 52)

3′-CACGGGCAMACCAGGCTTAGK-5′

L1

Forward:

(SEQ ID NO. 53)

5′-KCAGATGGCYTTGYGGCGTACTA-3′

Reverse:

(SEQ ID NO. 56)

3′-GGGGCRTYRCGYTGACAKGTAGT-5′

Reverse:

(SEQ ID NO. 57)

3′-GGCMGGSCKTTTAAGGCCTGGT-5′

α6:

E7

Forward:

(SEQ ID NO. 64)

5′-GCTCAGAGGAWGAGGATGAGG-3′

Reverse:

(SEQ ID NO. 69)

3′-GCCTTGTTGCRCASAGGGG-5′

Reverse:

(SEQ ID NO. 70)

3′-CGCAGAGTGGGCACGTTACT-5′

L1

Forward:

(SEQ ID NO. 71)

5′-TTGCAGATGGCGRYGTGGCG-3′

Reverse:

(SEQ ID NO. 72)

3′-CACCTAAAGGYTGDCCDCGGC-5′

α7:

E7

Forward:

(SEQ ID NO. 82)

5′-GACGRGMHGAACMACARCGTCAC-3′

Reverse:

(SEQ ID NO. 85)

3′-GTGWSTCCATAAACAGCWGCWGT-5′

Reverse:

(SEQ ID NO. 86)

3′-CACACCAMGGACACACAAAGGAC-5′

L1

Forward:

(SEQ ID NO. 87)

5′-GCGBTCTAGYGACARCAHGGTGT-3′

Forward:

(SEQ ID NO. 88)

5′-HCCTGCTATTGGKGARCAYTGGG-3′

Reverse:

(SEQ ID NO. 89)

3′-CCAGTGYTCYCCMATRGCRGGWA-5′

Reverse:

(SEQ ID NO. 90)

3′-TAGASCCACTDGGWGANGGRGAA-5′

α10:

E7

Forward:

(SEQ ID NO. 122)

5′-GCWCAYTWGGAATHGTGTGCCCC-3′

Forward:

(SEQ ID NO. 123)

5′-CSTGTAAMAACGCCATGAGAGGA-3′

Forward:

(SEQ ID NO. 124)

5′-CGCCATGAGAGGAMACAASCCA-3′

Reverse:

(SEQ. ID NO. 125)

3′-GGCACACDATTCCWARTGWGCCC-5′

Reverse:

(SEQ ID NO. 126)

3′-GGTTCGTASGTCRSTTGYTGTAC-5′

Reverse:

(SEQ ID NO. 127)

3′-GTGCACAGSYGGGRCACACWAYT-5′

L1

Forward:

(SEQ ID NO. 128)

5′-GARGCCACWGTSTACYTGCCTC-3′

Forward:

(SEQ ID NO. 129)

5′-ACAGATGTCTCTGTGGCGGC-3′

Reverse:

(SEQ ID NO. 130)

3′-GGATGNCCACTWAYRCCHACDCC-5′

Example 2: Quantifying E6 and E7 Reads Versus Other Viral Reads

A sample of cells is collected from the cervix using a spatula or small brush and put in a conservative solution. RNAs are extracted from the cells using standard procedure and polyA mRNAs are selected using standard procedures like using poly dT beads. Libraries are prepared using standard library preparation (RNA fragmentation and reverse transcription into double-stranded complementary DNA primed by random hexamer followed by adapter selection, or reverse transcription to single strand cDNA, ligation of cDNA and random amplification by phi 29 polymerase followed by fragmentation and adpaterligation). Alternatively RT-PCT is conducted using set of primers for E6 and E7 and at least one another late gene as described. After sequencing using several million reads of at least 100 nt, reads are mapped on a database of E6 and E7 genes: HPV genotypes expressing E6/E7 are identified. The other reads are mapped on the subset of genomes corresponding to the corresponding genotypes. Within each genotype, ratio of the number of E6/E7 reads to the reads mapped to at least one anther gene is calculated and compared to thresholds.

Example 3: Biological Samples

Two high grade lesions (HSIL) samples of the cervix from two donor women, hereinafter referred to as 117 and 119, were collected in PreservCyt medium (Hologic) and kept at room temperature for a couple of days. After homogenization, 1 mL aliquots were collected from the 20 μL total liquid medium for HPV genotyping (Papillocheck, Greiner Bio-One). Results of HPV typing are given in table 1. The remaining samples were centrifuged at 4,500×g for 10 min and the pellets were stabilized in 1 mL RNAProtect Cell (Qiagen) for storage at −80° C. before RNA extraction.

Example 4: HPV Database

Sixty four (64) reference sequences representing the entire HPV alpha genus were retrieved from the International Human Papillomavirus Reference Center (updated May 2014). Additional nine (9) sublineage sequences corresponding to HPV16, plus nine (9) sublineage sequences corresponding to HPV18 (described in Burk et al. Virology 2013) were added. The resulting eighty two (82) HPV genomes (listed in table 2) were aligned using ClustalW2 (default parameters) and the output file was analyzed using the Geneious software (Geneious 7.1.5, Biomatters Ldt).

Example 5: Design of HPV Reverse Transcription Primers

A dedicated strategy for the design of HPV reverse transcription (RT)-primers was set up with the goal to carry out a specific enrichment of HVP sequences during the reverse transcription step within a ballast of viral and non-viral RNA sequences. The overall approach consists in targeting the entire early and late populations of HPV transcripts starting from a limited number of specific RT primers. The design is achieved by taking advantage of the sequence shared by all early and late transcripts, located in the 5′ vicinity of the early and late polyA signals, respectively (FIG. 1). In order to minimize the number of RT primers required to cover all kind of alpha HPV, the degree of similarity between the 82 HPV genomes was taken into account. Additional criteria for the design of RT primers were as follows: (i) an overall good specificity of the primer aligned against all existing sequences databases (BLAST NCBI) with special attention paid in considering the 3′ part of the primer, (ii) a GC content around 50% (+1-12%), (iii) a melting temperature (Tm) >50° C. (assuming 0.2 μM primers and 50 mM salt), (iv) no T tracts, (v) no or low GC content in the 3′ part of the primer and (vi) no or limited number of putative secondary structures. This approach was implemented manually as a proof of principle to design one (1) RT-specific primer targeting the early transcripts of the 9 HPV16 sequences, one (1) RT-specific primer targeting the late transcripts of the 9 HPV16 sequences, two (2) RT-specific primers targeting the early transcripts of the 9 HPV18 sequences and two (2) RT-specific primers targeting the late transcripts of the 9 HPV18 sequences. The resulting six (6) RT-specific primers targeting both HPV16 and HPV18 sequences and including sublineages are given in table 3. HPSF-purified primers (0.01 μmop were ordered at Eurofins genomics (http://www.eurofinsgenomics.eu).

Example 6: RNA Extraction and Characterization

Total RNA from samples 117 and 119 were extracted using the PicoPure RNA isolation kit (Life Technologies), adding a DNAse treatment step directly on column (RNAse-free DNAse set, Qiagen) as recommended by the supplier. Elution was achieved in 30 μL elution buffer. Assessment of RNA quantity and quality was done with a Nanodrop 1000 (Thermo Scientist) and a Bioanalyzer 2100 using the RNA Nano chips (Agilent).

Example 7: Random Reverse Transcription

Random reverse transcription of total RNA was carried out using the SuperScript III First-Strand cDNA Synthesis kit (Invitrogen). Briefly, 8 μL of total RNA was used for template and the reaction was performed in the presence of 50 nM random hexamers (provided by Invitrogen), incubated 10 min at 25° C., 50 min at 50° C. and 5 min at 85° C. before a final RNAse H treatment 20 min at 37° C. The resulting cDNA were stored at −20° C.

Example 8: HPV-Specific Reverse Transcription

HPV-specific reverse transcription was carried out using the SuperScript III First-Strand cDNA Synthesis kit (Invitrogen) and primed with the HPV-specific RT-primers described above. Briefly, 8 μL of total RNA was used for template and reaction was performed with a 0.2 μM mixture of the 6 HPV-specific RT primers, incubated 50 min at 50° C. and 5 min at 85° C. before a final RNAse H treatment 20 min at 37° C. The resulting cDNA were stored at −20° C.

Example 9: Control PCR

The HPV16 E7 and the human cellular beta-actin (ACTB) genes were used as controls of the random and HPV-specific reverse-transcription steps, respectively. 1 μL of reverse-transcribed cDNA was used as PCR templates in 20 μL final volume, working with LightCycler DNA Master SybrGreen I reagents (Roche Diagnostics). 45 amplification cycles were achieved on a Light Cycler 480 (Roche) as follows: 95° C. 10 sec, 56° C. 10 sec, 72° C. 30 sec. Fusion curves and electrophoresis gels served for validation. A comparison of Ct values obtained by following either the random RT or the HPV RT protocol is given in table 4.

Example 10: Whole Transcriptome Random Amplification

cDNA were randomly amplified using the Multiple Displacement Amplification (MDA) protocol with phi29 polymerase and random hexamers (Whole Transcriptome Amplification, Qiagen). Phi 29 was UV-treated for one hour before use, in order to prevent any residual DNA contaminant.

Example 11: High Throughput Sequencing

Samples 117 (both random RT and HPV-specific RT) and 119 (both random RT and HPV-specific RT) were independently analyzed on two sequencing runs (300 bp paired-end sequencing, TruSeq PCR-free library prep, 600 cycle kit) on a MiSeq apparatus (Illumina). fastQ data were generated and QC tests done following standard procedures. Total numbers of sequencing reads per sample are summarized in table 5.

Example 12: Data Analysis

Quality-filtered reads were mapped to reference sequences using the following criteria: (i) alignment identity of at least 90% and (ii) Smith and Waterman score above 100. A selection of 10 human genes served as cellular controls (table 6). The analysis of the reads mapping HPV sequences relied on two strategies: first, at the genomic level, sequencing reads mapping HPV16 (NC_001526.2), HPV6 (HG793939.1) and HPV35 (JX129488.1) were count for each coding sequence (CDS), without adding any particular filter (table 7). In a second and more transcript-specific approach, reads mapping splice junctions of HPV16 were identified (table 8). This latter analysis was done for HPV16 only as a proof of principle, and relied on well-documented donor and acceptor splice sites, as described for example in Zheng et al. FrontBiosci 2006.

Example 13: Sequence Results

Following the random reverse-transcription protocol, the sequencing of patient 117 resulted in a total of 1,455 and 126 reads (over 34,977,682) that were successfully mapped to the HPV16 (NC_001526.2) and HPV35 (JX129488.1) genomes, respectively. 15 reads (over 39675490) were mapped to the HPV16 genome for the mono-infected patient 119. Following the alternative procedure with HPV-specific RT primers, the sequencing of patient 117 resulted in a total of 2033, 69 and 14 reads (over 28598603) for HPV16, HPV35 and HPV6 genomes, respectively. 6 reads (over 19383833) were mapped to the HPV16 genome for patient 119.

Two lines of analysis were conducted in order to characterize finely different populations of HPV reads. First, at the genomic level, reads mapping CDS regions were counted, giving a broad view of phenomena such as the early vs late genes equilibrium (table 7). In addition to that, we sought to characterize deeply specific HPV16 transcripts by taking advantage of well-documented donor and acceptor splice sites described for HPV16. This led us to define 11 spliced transcripts which can be associated unambiguously to one specific RNA event (table 8). Together, these two analysis showed that (i) HPV sequences are reachable using HTS, (ii) it is possible to perform a gene-by-gene reads counting at the genomic level, (iii) reads associated to specific splice junctions exist and can be characterized and counted as well, confirming essentially the detection of transcripts over possible artefacts introduced by residual HPV DNA, (iv) discrepancies exist between samples, between HPV genus and between HPV genes and transcripts patterns, which reflect probably the diversity of HPV infections.

Example 14: Examples of R Scores

These observations opened the possibly to define a score, referred to as R score, based on HPV CDS counts and/or specific transcripts within each genotype present in a given sample, to gain a fine molecular characterization of any individual HPV-positive samples. From this perspective, either one value or a combination of more than one ratio(s) could be considered. A non-restrictive list of R scores is given in table 9 in order to illustrate several possible combinations based either on CDS or specific transcripts. As an example, R scores based on a ratio E6 and/or E7 and/or E2 and/or L1 and/or L2 succeeded in generating high score values (highlighted) that should be associated of non- or lowly-productive HPV cycles typical of transformed cells. Of note, weighting coefficients such as αE6 and/or βE7 and/or γE2 and/or δL1 and/or εL2 can be added as parameters, independently, in order to better discriminate, for instance, low risk and high risk lesions.

Example 15: Random RT Vs HPV-Specific RT

As an alternative to the conventional random RT upstream of random amplification, we attempted to define and to use HPV-specific RT primers, with the ultimate goal to achieve a specific HPV enrichment over non-HPV sequences. Such targeted (semi-random) approach may prove extremely important in the perspective of reducing the depth sequencing (that is dependent on the ratio of HPV to non HPV sequences), increasing multiplexing, and reducing costs required before being able to use HTS as a screening test. Although the number of HPV reads remains roughly comparable between the random RT and the HPV-specific RT approaches, a marked difference was observed regarding cellular genes, as exemplified by both PCR (table 4, average ACTB ΔCt −3.01 for HPV RT compared to E7 ΔCt −0.56) and HTS results (table 6, average 3.3 fold reduction for HPV RT, after total reads number correction). In addition to that, HPV6 reads were detectable in the poly-infected sample 117 only when applying the HPV RT approach, thus recovering the results of the Papillocheck gold standard genotyping test. These results, albeit based on a limited number of experimental evidences, suggest a minima that our innovative HPV-specific reverse transcription approach coupled with random amplification is able to reduce the cellular and other non-HPV ballasts, without deteriorate the detection of specific HPV targets. Optimizations of the technique are now required to achieved a strong HPV enrichment and to afford linear quantification.

TABLE 1

Biological samples and associated HPV-genotyping

Sample
Year of birth
Lesion
HPV typing (Papillocheck)

117
1969
High grade (HSIL)
6, 16, 35

119
1986
High grade (HSIL)
16

TABLE 2

Seq
Virus
Genus
Species
GenBank
HR αHPV

index
name
name
name
ID
(x)

1
HPV2
Alpha
Alpha-4
X55964

2
HPV3
Alpha
Alpha-2
X74462

3
HPV6
Alpha
Alpha-10
X00203

4
HPV7
Alpha
Alpha-8
X74463

5
HPV10
Alpha
Alpha-2
X74465

6
HPV11
Alpha
Alpha-10
M14119

7
HPV13
Alpha
Alpha-10
X62843

8
HPV16
Alpha
Alpha-9
K02718

9
HPV16
Alpha
Alpha-9
AF536179

10
HPV16
Alpha
Alpha-9
HQ644236

11
HPV16
Alpha
Alpha-9
AF534061

12
HPV16
Alpha
Alpha-9
AF536180

13
HPV16
Alpha
Alpha-9
HQ644298

14
HPV16
Alpha
Alpha-9
AF472509

15
HPV16
Alpha
Alpha-9
HQ644257

16
HPV16
Alpha
Alpha-9
AY686579
x

17
HPV16
Alpha
Alpha-9
AF402678

18
HPV18
Alpha
Alpha-7
X05015
x

19
HPV18
Alpha
Alpha-7
AY262282

20
HPV18
Alpha
Alpha-7
EF202146

21
HPV18
Alpha
Alpha-7
EF202147

22
HPV18
Alpha
Alpha-7
EF202151

23
HPV18
Alpha
Alpha-7
GQ180787

24
HPV18
Alpha
Alpha-7
EF202155

25
HPV18
Alpha
Alpha-7
KC470225

26
HPV18
Alpha
Alpha-7
EF202152

27
HPV18
Alpha
Alpha-7
KC470229

28
HPV26
Alpha
Alpha-5
X74472

29
HPV27
Alpha
Alpha-4
X74473

30
HPV28
Alpha
Alpha-2
U31783

31
HPV29
Alpha
Alpha-2
U31784
x

32
HPV30
Alpha
Alpha-6
X74474

33
HPV31
Alpha
Alpha-9
J04353
x

34
HPV32
Alpha
Alpha-1
X74475

35
HPV33
Alpha
Alpha-9
M12732
X

36
HPV34
Alpha
Alpha-11
X74476

37
HPV35
Alpha
Alpha-9
X74477

38
HPV39
Alpha
Alpha-7
M62849

39
HPV40
Alpha
Alpha-8
X74478
x

40
HPV42
Alpha
Alpha-1
M73236

41
HPV43
Alpha
Alpha-8
AJ620205

42
HPV44
Alpha
Alpha-10
U31788

43
HPV45
Alpha
Alpha-7
X74479

44
HPV51
Alpha
Alpha-5
M62877
x

45
HPV52
Alpha
Alpha-9
X74481

46
HPV53
Alpha
Alpha-6
X74482

47
HPV54
Alpha
Alpha-13
U37488

48
HPV56
Alpha
Alpha-6
X74483

49
HPV57
Alpha
Alpha-4
X55965

50
HPV58
Alpha
Alpha-9
D90400

Si
HPV59
Alpha
Alpha-7
X77858
x

52
HPV61
Alpha
Alpha-3
U31793
x

53
HPV62
Alpha
Alpha-3
AY395706

54
HPV66
Alpha
Alpha-6
U31794

55
HPV67
Alpha
Alpha-9
D21208

56
HPV68
Alpha
Alpha-7
X67161
x

57
HPV69
Alpha
Alpha-5
AB027020

58
HPV70
Alpha
Alpha-7
U21941
x

59
HPV71
Alpha
Alpha-14
AB040456
x

60
HPV72
Alpha
Alpha-3
X94164

61
HPV73
Alpha
Alpha-11
X94165

62
HPV74
Alpha
Alpha-10
AF436130

63
HPV77
Alpha
Alpha-2
Y15175

64
HPV78
Alpha
Alpha-2
AB793779

65
HPV81
Alpha
Alpha-3
AJ620209

66
HPV82
Alpha
Alpha-5
AB027021
x

67
HPV83
Alpha
Alpha-3
AF151983

68
HPV84
Alpha
Alpha-3
AF293960
x

69
HPV85
Alpha
Alpha-7
AF131950

70
HPV86
Alpha
Alpha-3
AF349909

71
HPV87
Alpha
Alpha-3
AJ400628

72
HPV89
Alpha
Alpha-3
AF436128

73
HPV90
Alpha
Alpha-14
AY057438
x

74
HPV91
Alpha
Alpha-8
AF419318

75
HPV94
Alpha
Alpha-2
AJ620211

76
HPV97
Alpha
Alpha-7
DQ080080

77
HPV102
Alpha
Alpha-3
DQ080083

78
HPV106
Alpha
Alpha-14
DQ080082

79
HPV114
Alpha
Alpha-3
GQ244463

80
HPV117
Alpha
Alpha-2
GQ246950

81
HPV125
Alpha
Alpha-2
FN547152

82
HPV160
Alpha
Alpha-2
AB745694
x

TABLE 3

Primers used for HPV-specific

reverse transcription

Primer
Sequence
Length
%

SEQ

name
(5′->3′)
(bp)
GC
Tm
ID NO.

HPV16-
CAGCGGACGT
19
47
54.5
SEQ ID

early
ATTAATAGG

NO.153

HPV16-
TCATATTCCT
19
47
54.5
SEQ ID

late
CCCCATGTC

NO.154

HPV18-
AGGGGACGT

SEQ ID

early-
TATTACCAC
18
50
53.7
NO.155

pop1

HPV18-
CAGGGGACGT
19
47
54.5
SEQ ID

early-
TATTATCAC

NO.156

pop2

HPV18-
ATATTCCTCA
20
40
53.2
SEQ ID

late-
ACATGTCTGC

NO.157

pop1

HPV18-
CATATTCTTCA
21
38
54.0
SEQ ID

late-
ACATGTCTGC

NO.158

pop2

TABLE 4

Comparative Ct values obtained by PCR after random or HPV-specific

reverse transcription

Human ACTB
E7 HPV16

Random

Random

RT
HPV RT
ΔCt
RT
HPV RT
ΔCt

Sample 117
26.69
29.48
−2.79
37.23
37.35
−0.12

Sample 119
23.97
27.20
−3.23
35.25
36.25
−1.00

TABLE 5

Total number of sequencing reads

Total reads # (raw)
Quality Filtering

Sample 117 random RT
37,055,284
34,977,682

Sample 117 HPV RT
30,607,370
28,598,603

Sample 119 random RT
41,994,892
39,675,490

Sample 119 HPV RT
20,462,884
19,383,833

TABLE 6

Number of sequencing reads mapping human cellular genes (GRCh37) after random or

HPV-specific reverse transcription

ACTB
GAPDH
G6PD
HPRT1
RPLP0
GUSB
PPIA
KRT19
CDKN2A
MKI67P1
TOTAL

Sample 117 random RT
731
7
1
32
0
4
4
10
5
0
794

Sample 117 HPV RT
86
0
0
13
2
2
2
5
11
0
121

Sample 119 random RT
546
19
9
183
38
23
50
57
96
0
1,021

Sample 119 HPV RT
47
5
28
146
6
17
50
0
73
12
384

TABLE 7

Number of sequencing reads mapping HPV CDS

Sample 117
Sample 119

HPV
HPV16
HPV35
HPV6
HPV16

CDS
Rand. RT
HPV RT
Rand. RT
HPV RT
Rand. RT
HPV RT
Rand. RT
HPV RT

E6
162
121
0
0
0
0
6
2

E7
91
91
7
0
0
14
9
0

E1
585
862
30
7
0
13
3
3

E2
366
684
22
0
0
0
1
0

E4
29
33
0
0
0
0
0
0

E5
71
70
0
0
0
0
1
0

L2
248
211
12
11
0
0
0
0

L1
305
535
24
47
0
0
0
1

TABLE 8

Number of sequencing reads mapping spliced HPV16 transcripts

Genomic coordinates
Sample 117
Sample 119

of HPV16 splice sites
Number of reads at
Number of reads at

HPV16 splice
(NC_001526.2)
the splice junction
the splice junction

transcripts
Spl. Donor
Spl. Accep.
Rand. RT
HPV RT
Rand. RT
HPV RT

E6*I
226
409
33
25
0
0

E6*II
226
526
4
0
0
0

E6*III, E5
226
3358
0
0
0
0

E6*IV
226
2709
36
23
0
0

E6{circumflex over ( )}E7
226
742
0
0
0
0

E1C
880
2582
0
0
0
0

E1{circumflex over ( )}E4
880
3358
12
4
0
0

E2
880
2709
0
2
0
0

E2C
1302
3358
0
0
0
0

L1
3632
5639
0
0
0
0

L1*
1302
5639
0
0
0
0

Example 16: Summary of Examples 1-15

The method according to the present invention described in the Examples above comprises:

1. Extraction of viral RNAs (Example 6) from a biological sample (Example 3),
2. Reverse transcription of the RNAs into cDNA with random hexamers (Example 7) or primers specific for HPV (Example 8); the design of the primers being illustrated by Example 1 (consensus primer) and Example 5 (HPV16 and HPV18 specific primers). A cDNA quality control is carried out by quantitative PCR (Example 9).
3. Amplification of cDNA by MDA technology with random hexamers (Example 10) to generate a DNA sequence bank (Example 2),
4. High throughput sequencing of the DNA bank and generation of “sequencing reads” (Example 1.1),
5. Aligning reads (Example 12) with the sequences of the HPV genomes present in the database (Example 5). Two analytical strategies are possible (Example 12, results in Example 13):
- a. counting reads aligning with each CDS of interest, or
- b. enumeration of reads aligning only the known splice junctions of each CDS of interest;
6. Computing R score (Example 14) whose the different possible computings are ratios described in Table 9. The ratio is defined as the ratio between the number of reads generated for at least 2 genes described in the present patent application.

Example 17: Detection and Quantification of HPV16 and Human Transcripts

17.1 HPV Database

Sixty four (64) genomic sequences representing the HPV alpha genus were retrieved from the International Human Papillomavirus Reference Center (http://www.hpvcenter.se/index.html; updated May 2014). Additional nine (9) sublineage sequences corresponding to HPV16, plus nine (9) sublineage sequences corresponding to HPV18 (described in Burk et al. Virology 2013) were added. The resulting eighty two (82) HPV genomes are referred to as the αHPV database (Table 2 above). A subgroup of the αHPV database composed of sixteen (16) sequences (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82), corresponding to high risk or putative high risk Papillomaviruses, is referred to as the HR αHPV group.

17.2 Delineation of Transcription Maps for HR αHPV

For each sequences of the HR αHPV group, known and putative/predicted splice donor (SD) and splice acceptor (SA) sites were annotated. First, previously documented SD and SA sites for HPV16 and HPV18 were retrieved from Zheng et al. Front Biosci. 2006, Wang et al. Journal of Virology 2011 and Toots et al. PLoS ONE 2014 (FIG. 7, light grey). Based on the idea that virtually all spliced sites are analogous to those previously described for other papillomaviruses (Wang et al. Journal of Virology 2011), the annotation of SA and SD was then expanded to the entire αHPV group by direct analogy (FIG. 7, black numbers). In addition, SA/SD predictions were supported by online splice site prediction software.

17.3 Transcripts Database and Primers Design

A database of short (150 to 500 bp) sequences reconstructed 5′ from the splice donor site and 3′ from the splice acceptor site was generated, gathering each transcript, part of transcript, or putative transcript for each HR αHPV, and is referred to as the αHPV transcripts database. This database served as a basis for the design of PCR primers. More precisely, a pair of primers was defined for each transcript, part of transcript or putative transcript when possible, with the objective of encompassing the splice junction, as defined in FIG. 7 (the corresponding transcripts appear with suffix ‘sp_’ in Table 10).

TABLE 10

Primer pairs for HPV16 and human transcripts

SEQ

SEQ

Transcript

ID

ID

targeted
Forward primer
NO.
Reverse primer
NO.

Human
ACTB.E4E5
CCAGGTCATCACCATTGGCAAT
159
CGTACAGGTCTTTGCGGATGT
160

Transcripts
AKT1.E2EB
CCATGAGCGACGTGGCTATT
161
CTCACGTTGGTCCACATCCT
162

B2M.E1E2
CTGTGCTCGCGCTACTCT
163
CAACTTCAATGTCGGATGGATGAAAC
164

BCL2.E2E3
GTGGATGACTGAGTACCTGAACC
165
GGCCAAACTGAGCAGAGTCTT
166

BRAF.E11E12
CGGGACTCGAGTGATGATTGG
167
CTGAGGTGTAGGTGCTGTCA
168

CDH1.E10E11
CTCCTGAAAAGAGAGTGGAAGTGT
169
CCGGATTAATCTCCAGCCAGTT
170

CDKN2A.E1E2
AACGCACCGAATAGTTACGGT
171
ACGGGTCGGGTGAGAGT
172

CDKN2B.E1E2
CGGATCCCAACGGAGTCAA
173
ACCGGTCGGGTGAGAGT
174

ERBB2.E11E12
TCTTCCAGAACCTGCAAGTAATCC
175
GGTGGGTGTTATGGTGGATGA
176

FOS.E3E4
AGGAGAATCCGAAGGGAAAGGAATA
177
TCCTTCAGCAGGTTGGCAAT
178

GAPDH.E5E6
AGTCCACTGGCGTCTTCAC
179
TGATCTTGAGGCTGTTGTCATACTTC
180

GUSB.E10E11
GCGAGTATGGAGCAGAAACGA
181
AATTCCAAATGAGCTCTCCAACCA
182

HRAS.E2E3
CGGAATATAAGCTGGTGGTGGT
183
GCACGTCTCCCCATCAATGA
184

KRAS.E3E4
GTGCAATGAGGGACCAGTACA
185
CTACTAGGACCATAGGTACATCTTCAGA
186

KRT10.E3E4
GATGAGCTGACCCTGACCAA
187
GGCAGCATTCATTTCCACATTCAC
188

KRT14.E3E4
AGGAGCTGGCCTACCTGAA
189
CTTCTCATACTGGTCACGCATCT
190

KRT17.E1E2
AACACTGAGCTGGAGGTGAAG
191
CTGTAGCAGGATGTTGGCATTG
192

MET.E2E3
TGTGTGCATTCCCTATCAAATATGTCAA
193
GCGCTTCACAGCCTGATGA
194

MKI67.E6E7
CGTCGTGTCTCAAGATCTAGCTT
195
TGAGTCATCTGCGGTACTGTCT
196

MYC.E1E2
GCTTCTCTGAAAGGCTCTCCTT
197
AAATACGGCTGCACCGAGT
198

NOTCH1.E31E32
CCGACGCACAAGGTGTCTT
199
GTCGGCGTGTGAGTTGATGA
200

PCNA.E4E5
GACGGAGTGAAATTTTCTGCAAGT
201
GAAGTTCAGGTACCTCAGTGCAAA
202

PTEN.E8E9
AGCGTGCAGATAATGACAAGGAA
203
GATTTGACGGCTCCTCTACTGT
204

RB1.E22E23
CGGTCTTCATGCAGAGACTGA
205
GTGAAATATAGATGTTCCCTCCAGGAAT
206

RPLP0.E7E8
GACGGATTACACCTTCCCACTT
207
GACTCTTCCTTGGCTTCAACCTTA
208

STAT1.E18E19
CGATGGGCTCAGCTTTCAGA
209
ACAAAACCTCGTCCACGGAAT
210

TERT.E10E11
TCCTGCGTTTGGTGGATGAT
211
CCTCGTCTTCTACAGGGAAGTTCA
212

TOP2A.E21E22
TGGGTGGTCCTGCAAAATCC
213
ACATATTGATTTGGAGCCAGTTCTTCA
214

TP53.E4E5
CTGGCCCCTGTCATCTTCTG
215
CTTGGCCAGTTGGCAAAACAT
216

WNT1.E2E3
CTGGAACTGTCCCACTGCT
217
CAGGATTCGATGGAACCTTCTGA
218

HPV16
unsp_226_227
CACAGAGCTGCAAACAACTATACAT
219
CACATACAGCATATGGATTCCCATCTC
220

genomic
unsp_408_409
GGAACAACATTAGAACAGCAATACAACA
221
TGTCCAGATGTCTTTGCTTTTCTTCA
222

and
unsp_525_526
CGGTGGACCGGTCGATG
223
TCAGTTGTCTCTGGTTGCAAATCT
224

unspliced
unsp_741_742
CTCAGAGGAGGAGGATGAAATAGATG
225
CCATTAACAGGTCTTCCAAAGTACGA
226

transcripts
unsp_880_881
GGAATTGTGTGCCCCATCTGT
227
CATCCATTACATCCCGTACCCT
228

unsp_p997_998
GGTTTTATGTAGAGGCTGTAGTGGAA
229
TGTGCAGTAAACAACGCATGTG
230

unsp_1301_1302
GCGGGTATGGCAATACTGAAGT
231
TGGTGTTTGGCATATAGTGTGTCTTT
232

gen_1553_2056
ATCAACGTGTTGCGATTGGT
233
CTAATAGTAACACAACCATTCCCCATGA
234

unsp_p2307_2308
GAGGTGATTGGAAGCAAATTGTTATGT
235
CAGACCCTTGCAGAAATTTCATTAAACT
236

unsp_2580_2581
GGATGTAAAGCATAGACCATTGGTACA
237
GTTTTCGTCAAATGGAAACTCATTAGGA
238

unsp_2707_2708
CGGAAATCCAGTGTATGAGCTTAATGAT
239
TGACACACATTTAAACGTTGGCAAAG
240

unsp_3356_3357
CATGCGGGTGGTCAGGTAA
241
AAGGCGACGGCTTTGGTAT
242

unsp_3631_3632
GCTCACACAAAGGACGGATTAAC
243
CCAATGCCATGTAGACGACACT
244

gen_3883_4218
GCGTGCTTTTTGCTTTGCTTTG
245
CAGAGGCTGCTGTTATCCACAATA
246

unsp_4619_p4620
TGGGCCCTTCTGATCCTTCTAT
247
GGTCAGTGAAAGTGGGATTATTATGTGT
248

unsp_p5009_5010
CTGCTTTTGTAACCACTCCCACTA
249
CCTAGAGGTTAATGCTGGCCTATG
250

unsp_5408_p5409
CTTCACATGCAGCCTCACCTA
251
GGAATATTGTATGCACCACCAAAAGG
252

unsp_5636_5637
CCTATAGTTCCAGGGTCTCCACAA
253
ATCCGTGCTTACAACCTTAGATACTG
254

gen_5889_6779
GGATGACACAGAAAATGCTAGTGCTTA
255
CACCTGGATTTACTGCAACATTGG
256

unsp_7029_p7030
ACCTCCAGCACCTAAAGAAGATGA
257
GGTGTAGCTTTTCGTTTTCCTAATGTAA
258

HPV16
sp_226_409
CACAGAGCTGCAAACAACTATACAT
259
TGTCCAGATGTCTTTGCTTTTCTTCA
260

spliced
sp_226_526
CACAGAGCTGCAAACAACTATACAT
261
TCAGTTGTCTCTGGTTGCAAATCT
262

transcripts
sp_226_742
CACAGAGCTGCAAACAACTATACAT
263
CCATTAACAGGTCTTCCAAAGTACGA
264

sp_226_p1087
CACAGAGCTGCAAACAACTATACAT
265
CACTAAGTGGACTACCAAATACTTTCGT
266

sp_226_2581
CACAGAGCTGCAAACAACTATACAT
267
GTTTTCGTCAAATGGAAACTCATTAGGA
268

sp_226_2708
CACAGAGCTGCAAACAACTATACAT
269
TGACACACATTTAAACGTTGGCAAAG
270

sp_226_3357
CACAGAGCTGCAAACAACTATACAT
271
AAGGCGACGGCTTTGGTAT
272

sp_226_p4620
CACAGAGCTGCAAACAACTATACAT
273
GGTCAGTGAAAGTGGGATTATTATGTGT
274

sp_226_p5409
CACAGAGCTGCAAACAACTATACAT
275
GGAATATTGTATGCACCACCAAAAGG
276

sp_226_5637
CACAGAGCTGCAAACAACTATACAT
277
ATCCGTGCTTACAACCTTAGATACTG
278

sp_226_p7030
CACAGAGCTGCAAACAACTATACAT
279
GGTGTAGCTTTTCGTTTTCCTAATGTAA
280

sp_880_p1087
GGAATTGTGTGCCCCATCTGT
281
CACTAAGTGGACTACCAAATACTTTCGT
282

sp_880_2581
GGAATTGTGTGCCCCATCTGT
283
GTTTTCGTCAAATGGAAACTCATTAGGA
284

sp_880_2708
GGAATTGTGTGCCCCATCTGT
285
TGACACACATTTAAACGTTGGCAAAG
286

sp_880_3357
GGAATTGTGTGCCCCATCTGT
287
AAGGCGACGGCTTTGGTAT
288

sp_880_p4620
GGAATTGTGTGCCCCATCTGT
289
GTGTCTAGTGTAATAGTGTGTGATTTAT
290

TATGTGT

sp_880_p5409
GGAATTGTGTGCCCCATCTGT
291
GGTAATATTTGTTATTGCACCACCTAAAAGG
292

sp_880_5637
GGAATTGTGTGCCCCATCTGT
293
ATTCCGTTGTCTTTACTATACCTTTAGTA
294

TTACTTG

sp_880_p7030
GGAATTGTGTGCCCCATCTGT
295
GGTGTAGCTTTTTTTCGTTTTCLTAATGTTTAA
296

sp_p997_p1087
GGTTTTATGTAGAGGCTGTAGTGGAA
297
CACTAAGTGGACTACCAAATACTTTCGT
298

sp_p997_2581
GGTTTTATGTAGAGGCTGTAGTGGAA
299
GTTTTCGTCAAATGGAAACTCATTAGGA
300

sp_p997_2708
GGTTTTATGTAGAGGCTGTAGTGGAA
301
TGACACACATTTAAACGTTGGCAAAG
302

sp_p997_3357
GGTTTTATGTAGAGGCTGTAGTGGAA
303
AAGGCGACGGCTTTGGTAT
304

sp_p997_p4620
GGTTTTATGTAGAGGCTGTAGTGGAA
305
GGTCAGTGAAAGTGGGATTATTATGTGT
306

sp_p997_p5409
GGTTTTATGTAGAGGCTGTAGTGGAA
307
GGTAATATTTGTTATTGCACCACCTAAAAGG
308

sp_p997_5637
GGTTTTATGTAGAGGCTGTAGTGGAA
309
ATCCGTGTCTTTACTAACTCTTAGATACTG
310

sp_p997_p7030
GGTTTTATGTAGAGGCTGTAGTGGAA
311
GGTGTAGCTTTfTTCGTTTTTTCCTTTAA
312

TTGTTTAA

sp_1301_2581
GCGGGTATGGCAATACTGAAGT
313
GTTTTCGTCAAATGGAAACTCATTAGGA
314

sp_1301_2708
GCGGGTATGGCAATACTGAAGT
315
TGACACACATTTAAACGTTGGCAAAG
316

sp_1301_3357
GCGGGTATGGCAATACTGAAGT
317
AAGGCGACGGCTTTGGTAT
318

sp_1301_5637
GCGGGTATGGCAATACTGAAGT
319
ATCCGTGTCTTTACTAACCTTAGTATTACTG
320

sp_1301_p4620
GCGGGTATGGCAATACTGAAGT
321
GGTCAGTGAAAGTGGGATTATTATGTGT
322

sp_1301_p5409
GCGGGTATGGCAATACTGAAGT
323
GGTAATATTTGTATTGCACCACCAAAAGG
324

sp_1301_p7030
GCGGGTATGGCAATACTGAAGT
325
GGTGTAGCTTTTTTCGTTTTTTCCTAATGTAA
326

sp_p2307_2708
GAGGTGATTGGAAGCAAATTGTTATGT
327
TGACACACATTTAAACGTTGGCAAAG
328

sp_p2307_3357
GAGGTGATTGGAAGCAAATTGTTATGT
329
AAGGCGACGGCTTTGGTAT
330

sp_p2307_5637
GAGGTGATTGGAAGCAAATTGTTATGT
331
ATTCCGTGCTTTACTATACCTTTAGTATTACTG
332

sp_p2307_p4620
GAGGTGATTGGAAGCAAATTGTTATGT
333
GGTCAGTGAAAGTGGGATTATTATGTGT
334

sp_p2307_p5409
GAGGTGATTGGAAGCAAATTGTTATGT
335
GGAATATTGTATGCACCACCAAAAGG
336

sp_p2307_p7030
GAGGTGATTGGAAGCAAATTGTTATGT
337
GGTGTAGCTTTTTCGTTTTTTCCTTTAAT
338

TGTTTAA

sp_3631_5637
GCTCACACAAAGGACGGATTAAC
339
ATCCGTGCTTACAACCTTAGATACTG
340

sp_3631_p4620
GCTCACACAAAGGACGGATTAAC
341
GGTCAGTGAAAGTGGGATTATTATGTGT
342

sp_3631_p5409
GCTCACACAAAGGACGGATTAAC
343
GGAATATTGTATGCACCACCAAAAGG
344

sp_3631_p7030
GCTCACACAAAGGACGGATTAAC
345
GGTTGTTAGCTTTTTCGTTT
346

TTTTCCTTTAATTGTTTAA

sp_p5009_p5409
CTGCTTTTGTAACCACTCCCACTA
347
GGAATATTGTATGCACCACCAAAAGG
348

sp_p5009_5637
CTGCTTTTGTAACCACTCCCACTA
349
ATCCGTGCTTACAACCTTAGATACTG
350

sp_p5009_p7030
CTGCTTTTGTAACCACTCCCACTA
351
GGTGTAGCTTTTTTTCGTTTTCCTAATGTAA
352

HPV16-
fus_880_MYC_
GGAATTGTGTGCCCCATCTGT
353
CTGAGAAGCCCTGCCCTTC
354

human
001_exon1

fusion
fus_880_MYC_
GGAATTGTGTGCCCCATCTGT
355
AAATACGGCTGCACCGAGT
356

transcripts
001_exon2

fus_880_MYC_
GGAATTGTGTGCCCCATCTGT
357
GGTTGATTCCAGTACTTCTTGTACCTTTTG
358

001_exon3

fus_880_PVT1_
GGAATTGTGTGCCCCATCTGT
359
ATCATGATGGCTGTATGTGCCA
360

002_exon3

fus_880_PVT1_
GGAATTGTGTGCCCCATCTGT
361
CATTGTGTTTCCTACCTAGTCGTTTATT
362

004_exon1

fUS_880_PVT1_
GGAATTGTGTGCCCCATCTGT
363
TCTTTGCTCGCAGCTCGT
364

005_exon1

fus_2869_MYC_
AGTACAGACCTACGTGACCATATAGAC
365
CTGAGAAGCCCTGCCCTTC
366

001_exon1

fus_2869_MYC_
AGTACAGACCTACGTGACCATATAGAC
367
AAATACGGCTGCACCGAGT
368

001_exon2

fus_2869_MYC_
AGTACAGACCTACGTGACCATATAGAC
369
GGTGATCCAGALTClGACCTTTTG
370

001_exon3

fus_2869_PVT1_
AGTACAGACCTACGTGACCATATAGAC
371
ATCATGATGGCTGTATGTGCCA
372

002_exon3

fus_2869_PVT1_
AGTACAGACCTACGTGACCATATAGAC
373
CATGGTTCCACCAGCGTTATT
374

004_exon1

fus_2869_PVT1_
AGTACAGACCTACGTGACCATATAGAC
375
TCTTTGCTCGCAGCTCGT
376

005_exon1

In particular, the nearest neighbor splice sites have been taken into consideration in order to minimize risks of co-amplifying several spliced isoforms with a given couple of primers. Additional primers pairs were defined, when possible, to amplify the boundaries at the 5′-SD-genomic and genomic-SA-3′ positions (suffix ‘unsp_’) to allow for a better quantitative monitoring of concomitant spliced and/or genomic/unspliced transcription events and refine if necessary the description of transcripts equilibrium in the course of HPV infection. To complete this view and provide extra controls, primers were also designed within some HPV genomic regions lacking known SD/SA sites (suffix ‘gen_’), meaning that the detection of such sequences could result only from locally unspliced transcription or DNA contamination. A selection of human transcripts has been included in the design as well for normalization purposes and/or to support or improve a combination of human and/or HPV transcripts being able to discriminate low grade vs high grade lesions of the cervix. Of note, extra fusion transcripts (′fus_′) were investigated and primers were conceived following HPV breakpoint hypothesis in the context of HPV integration within the two human locus MYC and PTV1, as discussed for example in Lu et al. PLoS ONE 2014, Tang et al. Nature Communication 2013, Wentzensen et al. Oncogene 2002 or Peter et al. Oncogene 2006. In this case, forward primers were located 5′ of HPV breakpoints (see FIG. 7) and reverse primers designed within the targeted human exons, thus allowing for the detection and fine characterization of hybrid HPV-human transcripts.

17.4 Biological Samples and Cell Line

Two high grade lesions (HSIL) samples of the cervix from two donor women, hereinafter referred to as 610 and 729, were collected in PreservCyt medium (Hologic) and kept at room temperature for a couple of days. After homogenization, 1 mL aliquots were collected from the 20 μL total liquid medium for HPV genotyping (Papillocheck, Greiner Bio-One). Results of HPV typing are given in Table 11.

TABLE 11

HPV16 mono-infected samples from patients

Sample
Year of birth
Lesion
HPV typing (Papillocheck)

610
1985
High grade (HSIL)
16

729
1950
High grade (HSIL)
16

The remaining samples were centrifuged at 4,500×g for 10 min and the pellets were stored at −80° C. before RNA extraction. In addition, SiHa cells (HPV16 genomic integration) were cultured and harvested, providing another source of RNA.

17.5 RNA Extraction and Characterization

Total RNA from samples 610 and 729 were extracted using the PicoPure RNA isolation kit (Life Technologies), adding a DNAse treatment step directly on column (RNAse-free DNAse set, Qiagen) as recommended by the supplier. Elution was achieved in 30 μL elution buffer. Assessment of RNA quantity and quality was done with a Nanodrop 1000 (Thermo Scientist) and a Bioanalyzer 2100 using the RNA Nano chips (Agilent).

17.6 Random Reverse Transcription

Random reverse transcription of total RNA was carried out using the SuperScript III First-Strand cDNA Synthesis kit (Invitrogen). Briefly, 2 μL of total RNA was used for template and the reaction was performed in the presence of 50 nM random hexamers (provided by Invitrogen), incubated 10 min at 25° C., 60 min at 50° C. and 5 min at 85° C. before a final RNAse H treatment 20 min at 37° C. The resulting cDNA were immediately amplified using the multiplex approach described below.

17.7 Multiplex Amplification of Specific Transcripts

Amplification of HPV along with human transcripts was performed from the cDNA of samples 610, 729 and SiHa using a mixture of primers (appropriate for AmpliSeg™ technology; Life technologies) in a multiplex-manner, by a 20 cycles of amplification reaction. Following amplification, sequencing libraries were constructed (Life technologies) and validated on a Bioanalyzer 2100 before sequencing.

17.8 High Throughput Sequencing and Data Analysis

Samples 610, 729 and SiHa were sequenced on an Ion PGM apparatus using an Ion 118 chip (Life Technologies). FastQ data were generated and QC tests done following standard procedures. For each sample, sequencing reads were trimmed according to their Phred quality score then mapped to the HPV transcripts database using Bowtie 2 (Langmead et al. Nature Methods 2012). For spliced transcripts, alignments that did not encompass the splice junction were removed from the analysis. The number of reads for each sample is detailed in Table 12.

TABLE 12

Reads number for HPV16 and human transcripts

Transcript targeted
610
729
SiHa

Human
ACTB.E4E5
258790
81325
53371

transcripts
AKT1.E2E3
837
3517
1412

B2M.E1E2
73613
101287
14808

BCL2.E2E3
80
937
1

BRAF.E11E12
n.d.
5509
1447

CDH1.E10E11
n.d.
37368
2018

CDKN2A.E1E2
137
300
365

CDKN2B.E1E2
n.d.
57289
2958

ERBB2.E11E12
n.d.
73226
2159

FOS.E3E4
n.d.
8966
9397

GAPDH.E5E6
79755
52860
70892

GUSB.E10E11
137
226
112

HRAS.E2E3
65
700
181

KRAS.E3E4
n.d.
1006
474

KRT10.E3E4
374
412
0

KRT14.E3E4
38273
6019
86442

KRT17.E1E2
n.d.
21485
260630

MET.E2E3
n.d.
1361
308

MKI67.E6E7
1
4
1210

MYC.E1E2
0
112
223

NOTCH1.E31E32
n.d.
15301
1420

PCNA.E4E5
116
609
1262

PTEN.E8E9
n.d.
1568
1392

RB1.E22E23
n.d.
2807
2571

RPLP0.E7E8
32454
38380
5104

STAT1.E18E19
n.d.
12373
7993

TERT.E10E11
n.d.
0
628

TOP2A.E21E22
8
15
4267

TP53.E4E5
n.d.
11798
5551

WNT1.E2E3
n.d.
0
0

HPV16 genomic
unsp_226_227
n.d.
0
9180

and unspliced
unsp_408_409
61
0
18

transcripts
unsp_525_526
1122
2
2871

unsp_741_742
2102
2
1651

unsp_880_881
n.d.
0
1079

unsp_p997_998
1610
0
383

unsp_1301_1302
140
2
592

gen_1553_2056
1740
1
297

unsp_p2307_2308
7923
1
608

unsp_2580_2581
11800
1
881

unsp_2707_2708
25162
2
828

unsp_3356_3357
2996
0
0

unsp_3631_3632
10497
2
0

gen_3883_4218
1661
0
2

unsp_4619_p4620
2685
0
0

unsp_p5009_5010
1619
1
0

unsp_5408_p5409
1690
0
1

unsp_5636_5637
3047
0
0

gen_5889_6779
1356
0
0

unsp_7029_p7030
5794
1
0

HPV16 spliced
sp_226_409
n.d.
7
905

transcripts
sp_226_526
n.d.
0
568

sp_226_742
n.d.
0
39

sp_226_p1087
n.d.
0
0

sp_226_2581
n.d.
0
0

sp_226_2708
n.d.
0
3

sp_226_3357
n.d.
0
0

sp_226_p4620
n.d.
0
0

sp_226_p5409
n.d.
0
0

sp_226_5637
n.d.
0
0

sp_226_p7030
n.d.
0
0

sp_880_p1087
n.d.
0
0

sp_880_2581
0
0
4

sp_880_2708
29
0
92

sp_880_3357
11874
2
0

sp_880_p4620
0
0
0

sp_880_p5409
0
0
0

sp_880_5637
0
0
0

sp_880_p7030
0
0
0

sp_p997_p1087
n.d.
0
0

sp_p997_2581
0
0
0

sp_p997_2708
0
0
0

sp_p997_3357
0
0
0

sp_p997_p4620
0
0
0

sp_p997_p5409
0
0
0

sp_p997_5637
0
0
0

sp_p997_p7030
0
0
0

sp_1301_2581
0
0
0

sp_1301_2708
0
0
0

sp_1301_3357
0
0
0

sp_1301_5637
0
0
0

sp_1301_p4620
0
0
0

sp_1301_p5409
0
0
0

sp_1301_p7030
0
0
0

sp_p2307_2708
0
0
0

sp_p2307_3357
0
0
0

sp_p2307_5637
0
0
0

sp_p2307_p4620
0
0
0

sp_p2307_p5409
0
0
0

sp_p2307_p7030
0
0
0

sp_3631_5637
30
0
0

sp_3631_p4620
0
0
0

sp_3631_p5409
0
0
0

sp_3631_p7030
0
0
0

sp_p5009_p5409
0
0
0

sp_p5009_5637
0
0
0

sp_p5009_p7030
0
0
0

HPV16-human
fus_880_MYC_001_exon1
0
0
0

fusion
fus_880_MYC_001_exon2
0
0
0

transcripts
fus_880_MYC_001_exon3
0
0
0

fus_880_PVT1_002_exon3
0
0
0

fus_880_PVT1_004_exon1
0
0
0

fus_880_PVT1_005_exon1
0
0
0

fus_2869_MYC_001_exon1
0
0
0

fus_2869_MYC_001_exon2
0
0
0

fus_2869_MYC_001_exon3
0
0
0

fus_2869_PVT1_002_exon3
0
0
0

fus_2869_PVT1_004_exon1
0
0
0

fus_2869_PVT1_005_exon1
0
0
0

17.9 Multiplex Amplification and Quantification of HPV16 Transcripts

As a proof of principle, it was seeked to discriminate 47 spliced transcripts (′sp_′), 16 unspliced transcripts (′unsp_′), 3 genomic transcripts (′gen_′), 12 putative HPV-human fusion transcripts (′fus_′), plus additional 30 human transcripts, from mono-infected HPV16 samples (samples 610 and 729) and SiHa cells. To ensure amplification specificity, the design has been checked for its lack of cross-match against the HPV database in addition to the human genome and transcripts databases. Primers are detailed in Table 10. Reads number following QC, mapping and validation of the splice junction are detailed in Table 12.

17.10 Results: Detection and Quantification of HPV16 and Human Transcripts

The experiment showed that (i) specific human transcripts, as internal and/or normalization controls, were detected in samples 610, 729 and SiHa with expression levels varying between transcripts and from one sample to another, thus validating the integrity of starting RNA material and the effectiveness of subsequent multiplex amplification steps (ii) specific spliced (′sp_′) and unspliced (′unsp_′) HPV16 transcripts were successfully detected and characterized in samples 610, 729 and SiHa, albeit in a variable proportion between samples, supporting the quantitative variations of specific HPV16 transcripts or transcription events between biological samples, (iii) in particular, sample SiHa exhibited no or rare genomic (′gen_′), unspliced (′unsp_′) and spliced (′sp_′) transcripts reads beyond genomic position 3356, which appeared consistent with the loss of viral late genes following HPV16 integration into the genome of SiHa cells, and (iv) it thus demonstrated the capability of the method to accurately differentiate between non-replicative, integrative HPV16 infection stages often associated with higher levels of E6/E7 transcripts (in this particular case sp_226_409, sp_226_526 and sp_226_742, see Table 12), from other anterior, HPV16-induced transformation and/or proliferation steps which usually imply transcription of the E2 and/or L1 and/or L2 genes (see as an example sp_880_3357 in Table 12). Consequently, specific HPV16 spliced transcripts and/or HPV16 unspliced transcripts and/or HPV16 genomic transcripts and/or HPV16-human fusion transcripts can be weighted to compute a score, or score ratio, discriminating different stages of interaction of HPV16 with infected cells, in particular the early vs late stages of HPV16 cycle, and/or the integrative vs non integrative forms of the HPV16 genome into infected cells, which are events associated to cell transformation. More generally, these results suggest that the method can be extended and applied to all HR αHPV.

17.11 Examples of R Scores

These observations reinforce the possibly to define a score, referred to as R score, based on specific HPV transcripts counts as a molecular marker of any individual HPV-positive samples. From this perspective, either one value or a combination of more than one ratio(s) could be used as a marker of the viral-cell interactions that shapes the transformation process. A non-restrictive list of R scores is given in Table 13 in order to illustrate several possible combinations based on specific HPV16 transcripts.

TABLE 13

Examples of R scores

R scores (examples)
610
729
SiHa

sp_226_409/sp_880_2708
n.d.
+∞
9.83

sp_880_2581/sp_3631_5637
0
n.a.
+∞

sp_880_2708/sp_3631_5637
0.96
n.a.
+∞

sp_880_3357/sp_3631_5637
395.8
+∞
n.a.

unsp_741_742/unsp_p5009_5010
1.29
2
+∞

As an example, R scores based on a ratio sp_226_409/sp_880_2708 and/or sp_880_2581/sp_3631_5637 and/or sp_880_2708/sp_3631_5637 and/or sp_880_3357/sp_3631_5637 and/or unsp_741_742/unsp_p5009_5010 succeeded in generating high score values (e.g.: +∞) that are associated with non- or lowly-productive HPV cycles typical of transformed cells. Of note, weighting coefficients such as α(sp_226_409/sp_880_2708) and/or β(sp_880_2581/sp_3631_5637) and/or γ(sp_880_2708/sp_3631_5637) and/or δ(sp_880_3357/sp_3631_5637) and/or ε(unsp_741_742/unsp_p5009_5010) can be added as parameters, independently, in order to better discriminate, for instance, low risk and high risk lesions.

17.12 Extension of the Method to the HR αHPV Group

The method was extended to the entire HR αHPV group (i.e.: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82) based on the transcription map described in FIG. 7. Primers resulting from this improved design are listed in Table 14.

TABLE 14

Primer pairs for HR aHPV group and human transcripts

Virus
Transcript
Forward primer
SEQ
Reverse primer
SEQ

name
targeted
(5′-3′)
ID NO
(5'-3')
ID NO

HVP16
16_fus_2869_
AGTACAGACCTACGTGACCATA
377
TCTTTGCTCGCAGCTCGT
378

PVT1_005_exon1
TAGAC

HVP16
16_sp_1301_2581
GCGGGTATGGCAATACTGAAGT
379
GTTTTCGTCAAATGGAAACTCAT
380

TAGGA

HVP16
16_sp_226_742
CACAGAGCTGCAAACAACTATACAT
381
CCATTAACAGGTCTTCCAAAGTA
382

CGA

HVP16
16_unsp_525_526
CGGTGGACCGGTCGATG
383
TCAGTTGTCTCTGGTTGCAAATC
384

T

HVP16
16_unsp_1301_1302
GCGGGTATGGCAATACTGAAGT
385
TGGTGTTTGGCATATAGTGTGTC
386

TTT

HVP16
16_sp_880_2581
GGAATTGTGTGCCCCATCTGT
387
GTTTTCGTCAAATGGAAACTCAT
388

TAGGA

HVP16
16_fus_2869_
AGTACAGACCTACGTGACCATA
389
AAATACGGCTGCACCGAGT
390

MYC_001_exon2
TAGAC

HVP16
16_fus_3619_
GCTCACACAAAGGACGGATTAAC
391
TCTTTGCTCGCAGCTCGT
392

PVT1_005_exon1

HVP16
16_fus_880_
GGAATTGTGTGCCCCATCTGT
393
TCTTTGCTCGCAGCTCGT
394

PVT1_005_exon1

HVP16
16_unsp_226_227
CACAGAGCTGCAAACAACTATACAT
395
CACATACAGCATATGGATTCCCA
396

TCTC

HVP16
16_sp_226_409
CACAGAGCTGCAAACAACTATACAT
397
TGTCCAGATGTCTTTGC
398

TTTTCTTCA

HVP16
16_sp_880_3357
GGAATTGTGTGCCCCATCTGT
399
AAGGCGACGGCTTTGGTAT
400

HVP16
16_unsp_2580_2581
GGATGTAAAGCATAGACCATTG
401
GTTTTCGTCAAATGGAA
402

GTACA

ACTCATTAGGA

HVP16
16_fus_2869_
AGTACAGACCTACGTGACCATA
403
GGTGATCCAGACTCTGACC
404

MYC_001_exon3
TAGAC

TTTTG

HVP16
16_fus_2869_
AGTACAGACCTACGTGACCATA
405
ATCATGATGGCTGTATGTGCCA
406

PVT1_002_exon3
TAGAC

HVP16
16_unsp_3631_3632
GCTCACACAAAGGACGGATTAAC
407
CCAATGCCATGTAGACGACACT
408

HVP16
16_fus_3619_
GCTCACACAAAGGACGGATTAAC
409
CATGGTTCCACCAGCGTTATT
410

PVT1_004_exon1

HVP16
16_fus_880_
GGAATTGTGTGCCCCATCTGT
411
GGTGATCCAGACTCTGAC
412

MYC_001_exon3

CTTTTG

HVP16
16_sp_226_2581
CACAGAGCTGCAAACAACTATACAT
413
GTTTTCGTCAAATGGAAACT
414

CATTAGGA

HVP16
16_sp_1301_2708
GCGGGTATGGCAATACTGAAGT
415
TGACACACATTTAAACGTTG
416

GCAAAG

HVP16
16_fus_880_
GGAATTGTGTGCCCCATCTGT
417
ATCATGATGGCTGTATGTGCCA
418

PVT1_002_exon3

HVP16
16_sp_3631_5637
GCTCACACAAAGGACGGATTAAC
419
ATCCGTGCTTACAACCTTAG
420

ATACTG

HVP16
16_unsp_3356_3357
CATGCGGGTGGTCAGGTAA
421
AAGGCGACGGCTTTGGTAT
422

HVP16
16_unsp_5636_5637
CCTATAGTTCCAGGGTCTCCACAA
423
ATCCGTGCTTACAACCTTAG
424

ATACTG

HVP16
16_fus_880_PVT1_
GGAATTGTGTGCCCCATCTGT
425
CATGGTTCCACCAGCGTTATT
426

004_exon1

HVP16
16_fus_2869_
AGTACAGACCTACGTGACCATA
427
CTGAGAAGCCCTGCCCTTC
428

MYC_001_exon1
TAGAC

HVP16
16_unsp_2707_2708
CGGAAATCCAGTGTATGAGCTT
429
TGACACACATTTAAACGTTG
430

AATGAT

GCAAAG

HVP16
16_sp_226_3357
CACAGAGCTGCAAACAACTATACAT
431
AAGGCGACGGCTTTGGTAT
432

HVP16
16_fus_3619_
GCTCACACAAAGGACGGATTAAC
433
CTGAGAAGCCCTGCCCTTC
434

MYC_001_exon1

HVP16
16_sp_1301_3357
GCGGGTATGGCAATACTGAAGT
435
AAGGCGACGGCTTTGGTAT
436

HVP16
16_sp_880_5637
GGAATTGTGTGCCCCATCTGT
437
ATCCGTGCTTACAACCTTA
438

GATACTG

HVP16
16_unsp_741_742
CTCAGAGGAGGAGGATGAAATAGATG
439
CCATTAACAGGTCTTCCAAA
440

GTACGA

HVP16
16_fus_880_MYC_
GGAATTGTGTGCCCCATCTGT
441
AAATACGGCTGCACCGAGT
442

001_exon2

HVP16
16_fus_3619_MYC_
GCTCACACAAAGGACGGATTAAC
443
GGTGATCCAGACTCTGACC
444

001_exon3

TTTTG

HVP16
16_fus_880_MYC_
GGAATTGTGTGCCCCATCTGT
445
CTGAGAAGCCCTGCCCTTC
446

001_exon1

HVP16
16_unsp_408_409
GGAACAACATTAGAACAGCAAT
447
TGTCCAGATGTCTTTGCTT
448

ACAACA

TTCTTCA

HVP16
16_unsp_880_881
GGAATTGTGTGCCCCATCTGT
449
CATCCATTACATCCCGTACCCT
450

HVP16
16_fus_3619_
GCTCACACAAAGGACGGATTAAC
451
ATCATGATGGCTGTATGTGCCA
452

PVT1_002_exon3

HVP16
16_sp_226_2708
CACAGAGCTGCAAACAACTATACAT
453
TGACACACATTTAAACGTT
454

GGCAAAG

HVP16
16_sp_880_2708
GGAATTGTGTGCCCCATCTGT
455
TGACACACATTTAAACGTTGGCA
456

AAG

HVP16
16_fus_3619_
GCTCACACAAAGGACGGATTAAC
457
AAATACGGCTGCACCGAGT
458

MYC_001_exon2

HVP16
16_sp_226_526
CACAGAGCTGCAAACAACTATACAT
459
TCAGTTGTCTCTGGTTGC
460

AAATCT

HVP16
16_fus_2869_
AGTACAGACCTACGTGACCATATAGAC
461
CATGGTTCCACCAGCGTTATT
462

PVT1_004_exon1

HVP16
16_gen_3881_4212
CGTGCTTTTTGCTTTGCTTTGT
463
GAGGCTGCTGTTATCCACAATA
464

GTAAT

HVP16
16_gen_5887_7259
CCTGTGTAGGTGTTGAGGTAGGT
465
TCTATTATCCACACCTGCA
466

TTTGCT

HVP16
16_gen_1551_2331
AACGTGTTGCGATTGGTGTATTG
467
CATTCCCCATGAACATGCTAAAC
468

TTTG

HVP16
16_gen_7266_7904
CCAGGCCCATTTTGTAGCTT
469
AGGTCAGGAAAACAGGGATTTG
470

G

HVP18
18_unsp_2650_2651
CTAAAATGTCCTCCAATACTACT
471
GTCATTTATTTCATATAC
472

AACCACAA

TGGATTGCCA

HVP18
18_sp_
TGCATCCCAGCAGTAAGCAA
473
GTCATTTATTTCATATA
474

929_2651

CTGGATTGCCA

HVP18
18_unsp_
GGATTGGACACTGCAAGACACA
475
CCCATGCTACATAGGTCATACAA
476

3165_3166

TTGTC

HVP18
18_sp_
GGATTGGACACTGCAAGACACA
477
ACGTCTGGCCGTAGGTCT
478

3165_3465

HVP18
18_unsp_
CAGAGGAAGAAAACGATGAAATA
479
AGAAACAGCTGCTGGAATGCT
480

790_791
GATGG

HVP18
18_unsp_
TCCTAAGAAACGTAAACGTGTTCCC
481
GTATTTACAACTCTTGCCACAGA
482

5612_5613

AGGA

HVP18
18_sp_
TCAGATAGTGGCTATGGCTGTTCT
483
GTCATTTATTTCATATAC
484

1357_2651

TGGATTGCCA

HVP18
18_fus_3684_
CAGCTACACCTACAGGCAACAA
485
GGTGATCCAGACTCTGAC
486

MYC_001_exon3

CTTTTG

HVP18
18_fus_
AATGACAGTAAAGACATAGACAG
487
AAATACGGCTGCACCGAGT
488

2943_MYC_
CCAAA

001_exon2

HVP18
18_sp_233_3465
TTCACTGCAAGACATAGAAATAA
489
ACGTCTGGCCGTAGGTCT
490

CCTGT

HVP18
18_fus_3684_
CAGCTACACCTACAGGCAACAA
491
ATCATGATGGCTGTATGTGCCA
492

PVT1_002_

exon3

HVP18
18_sp_
TCAGATAGTGGCTATGGCTGTTCT
493
GGTTTCCTTCGGTGTCTGCAT
494

1357_2779

HVP18
18_sp_
CAGCTACACCTACAGGCAACAA
495
TCAGGTAACTGCACCCTA
496

3696_5776

AATACTCTAT

HVP18
18_fus_
AATGACAGTAAAGACATAGACA
497
CATGGTTCCACCAGCGTTATT
498

2943_PVT1_
GCCAAA

004_exon1

HVP18
18_fus_3684_
CAGCTACACCTACAGGCAACAA
499
AAATACGGCTGCACCGAGT
500

MYC_001_

exon2

HVP18
18_fus_
CAGCTACACCTACAGGCAACAA
501
TCTTTGCTCGCAGCTCGT
502

3684_PVT1_005_

exon1

HVP18
18_unsp_
GCATATTTTATCATGCTGGCAGC
503
TCAGGTAACTGCACCCTAA
504

5775_5776
TCTA

ATACTCTAT

HVP18
18_sp_929_5613
TGCATCCCAGCAGTAAGCAA
505
GTATTTACAACTCTTGCCA
506

CAGAAGGA

HVP18
18_fus_929_
TGCATCCCAGCAGTAAGCAA
507
TCTTTGCTCGCAGCTCGT
508

PVT1_005_exon1

HVP18
18_fus_2943_
AATGACAGTAAAGACATAGACAG
509
ATCATGATGGCTGTATGTGCCA
510

PVT1_002_exon3
CCAAA

HVP18
18_sp_1357_3465
TCAGATAGTGGCTATGGCTGTTCT
511
ACGTCTGGCCGTAGGTCT
512

HVP18
18_fus_929_
TGCATCCCAGCAGTAAGCAA
513
AAATACGGCTGCACCGAGT
514

MYC_001_

exon2

HVP18
18_fus_929_
TGCATCCCAGCAGTAAGCAA
515
CTGAGAAGCCCTGCCCTTC
516

MYC_001_

exon1

HVP18
18_fus_2943_
AATGACAGTAAAGACATAGACA
517
TCTTTGCTCGCAGCTCGT
518

PVT1_005_exon1
GCCAAA

HVP18
18_fus_2943_
AATGACAGTAAAGACATAGAC
519
GGTGATCCAGACTCTGAC
520

MYC_001_exon3
AGCCAAA

CTTTTG

HVP18
18_sp_233_416
TTCACTGCAAGACATAGAAATA
521
CCCAGCTATGTTGTGAAATCGT
522

ACCTGT

HVP18
18_fus_929_
TGCATCCCAGCAGTAAGCAA
523
CATGGTTCCACCAGCGTTATT
524

PVT1_004_exon1

HVP18
18_unsp_1357_1358
TCAGATAGTGGCTATGGCTGTTCT
525
CCGTTGTCTATAGCCTCCGT
526

HVP18
18_sp_3696_5613
CAGCTACACCTACAGGCAACAA
527
GTATTTACAACTCTTGCCA
528

CAGAAGGA

HVP18
18_sp_929_2779
TGCATCCCAGCAGTAAGCAA
529
GGTTTCCTTCGGTGTCTGCAT
530

HVP18
18_sp_929_3465
TGCATCCCAGCAGTAAGCAA
531
ACGTCTGGCCGTAGGTCT
532

HVP18
18_unsp_233_234
TTCACTGCAAGACATAGAAATA
533
CTATACATTTATGGCATGCA
534

ACCTGT

GCATGG

HVP18
18_unsp_415_416
TCAGACTCTGTGTATGGAGACACAT
535
CCCAGCTATGTTGTGAAATCGT
536

HVP18
18_fus_2943_
AATGACAGTAAAGACATAGACAG
537
CTGAGAAGCCCTGCCCTTC
538

MYC_001_exon1
CCAAA

HVP18
18_fus_929_
TGCATCCCAGCAGTAAGCAA
539
GGTGATCCAGACTCTGACCTTTT
540

MYC_001_exon3

G

HVP18
18_sp_233_791
TTCACTGCAAGACATAGAAATA
541
AGAAACAGCTGCTGGAATGCT
542

ACCTGT

HVP18
18_fus_3684_
CAGCTACACCTACAGGCAACAA
543
CATGGTTCCACCAGCGTTATT
544

PVT1_004_exon1

HVP18
18_sp_929_5776
TGCATCCCAGCAGTAAGCAA
545
TCAGGTAACTGCACCCTAAATAC
546

TCTAT

HVP18
18_fus_929_
TGCATCCCAGCAGTAAGCAA
547
ATCATGATGGCTGTATGTGCCA
548

PVT1_002_exon3

HVP18
18_sp_233_2779
TTCACTGCAAGACATAGAAATA
549
GGTTTCCTTCGGTGTCTGCAT
550

ACCTGT

HVP18
18_sp_233_2651
TTCACTGCAAGACATAGAAATA
551
GTCATTTATTTCATATACT
552

ACCTGT

GGATTGCCA

HVP18
18_fus_3684_
CAGCTACACCTACAGGCAACAA
553
CTGAGAAGCCCTGCCCTTC
554

MYC_001_exon1

HVP18
18_unsp_929_930
TGCATCCCAGCAGTAAGCAA
555
CTCGTCATCTGATATTACAT
556

CTCCTGTT

HVP18
18_sp_3786_5776
CGAAAACATAGCGACCACTATA
557
TCAGGTAACTGCACCCTAAATAC
558

GAGAT

TCTAT

HVP18
18_unsp_3464_3465
TGACGACACGGTATCCGCTA
559
ACGTCTGGCCGTAGGTCT
560

HVP18
18_unsp_3786_3787
CGAAAACATAGCGACCACTATA
561
TTGTACACTATCTGGAATTGCAA
562

GAGAT

CAGT

HVP18
18_unsp_3696_3697
CAGCTACACCTACAGGCAACAA
563
GTCGCTATGTTTTCGCAATCTGT
564

A

HVP18
18_gen_1607_2401
TGGAGTAAACCCAACAATAGCAGAAG
565
CATTTGTAACGCAACAGGGCTA
566

AT

HVP18
18_sp_3786_5613
CGAAAACATAGCGACCACTATA
567
GTATTTACAACTCTTGCCACAGA
568

GAGAT

AGGA

HVP18
18_gen_7284_7857
CGCCCTAGTGAGTAACAACTGTATTT
569
GGAGGATTGTAGGATAAAATGG
570

ATGCT

HVP18
18_gen_6026_7277
GAGGACGTTAGGGACAATGTGT
571
CCCTGTGATAAAGGACGCGATT
572

T

HVP18
18_gen_3946_4234
CGTATGCATGGGTATTGGTATTTGTG
573
CATGTATATGCAATAGTAACATG
574

GGCAA

HVP31
31_unsp_5551_5552
GCCACAAGTGTCTATTTTTGTTGATG
575
TTTAGACACTGGGACAGGTGGT
576

A

HVP31
31_unsp_739_740
CAGATGAGGAGGATGTCATAGACAGT
577
CATTAACAGCTCTTGCAA
578

TATGCGAATA

HVP31
31_fus_3578_
CAGCTGCATGCACAAACCA
579
GGTGATCCAGACTCTGACC
580

MYC_001_exon3

TTTTG

HVP31
31_fus_2807_MYC_
CAACGTTTAAATGTGTGTCAGGA
581
AAATACGGCTGCACCGAGT
582

001_exon2
CAAA

HVP31
31_fus_2807_PVT1_
CAACGTTTAAATGTGTGTCAGGA
583
CATGGTTCCACCAGCGTTATT
584

004_exon1
CAAA

HVP31
31_fus_3578_PVT1_
CAGCTGCATGCACAAACCA
585
CATGGTTCCACCAGCGTTATT
586

004_exon1

HVP31
31_sp_3590_5552
CAGCTGCATGCACAAACCA
587
TTTAGACACTGGGACAGGTGGT
588

A

HVP31
31_fus_2807_
CAACGTTTAAATGTGTGTCAGGA
589
ATCATGATGGCTGTATGTGCCA
590

PVT1_002_exon3
CAAA

HVP31
31_unsp_1296_1297
GCGGGTATGGCAATACTGAAGT
591
TGGAGTTTCATTCTCTCGTT
592

CACTATG

HVP31
31_sp_1296_2646
GCGGGTATGGCAATACTGAAGT
593
CGTTGAGAAAGAGTCTCCATCG
594

TTTT

HVP31
31_sp_230_3295
CGGCATTGGAAATACCCTACGAT
595
GAATTCGATGTGGTGGTGTTGTT
596

G

HVP31
31_fus_2807_
CAACGTTTAAATGTGTGTCAGGA
597
CTGAGAAGCCCTGCCCTTC
598

MYC_001_
CAAA

exon1

HVP31
31_sp_230_740
CGGCATTGGAAATACCCTACGAT
599
CATTAACAGCTCTTGCAATATGC
600

GAATA

HVP31
31_fus_2807_
CAACGTTTAAATGTGTGTCAGGA
601
GGTGATCCAGACTCTGACCTTTT
602

MYC_001_
CAAA

G

exon3

HVP31
31_fus_877_
AATCGTGTGCCCCAACTGT
603
CATGGTTCCACCAGCGTTATT
604

PVT1_004_

exon1

HVP31
31_unsp_2645_
CTGGTGGTTTTTACATTTCCAAA
605
CGTTGAGAAAGAGTCTCCATCG
606

2646
TCCAT

TTTT

HVP31
31_sp_877_5552
AATCGTGTGCCCCAACTGT
607
TTTAGACACTGGGACAGGTGGT
608

A

HVP31
31_sp_230_413
CGGCATTGGAAATACCCTACGAT
609
TTTTCTTCTGGACACAACGGTCT
610

T

HVP31
31_sp_1296_2518
GCGGGTATGGCAATACTGAAGT
611
AATGTAAAAACCACCAGTCTGCT
612

ATGTA

HVP31
31_sp_230_2646
CGGCATTGGAAATACCCTACGAT
613
CGTTGAGAAAGAGTCTCCATCG
614

TTTT

HVP31
31_sp_877_2518
AATCGTGTGCCCCAACTGT
615
AATGTAAAAACCACCAGTCTGCT
616

ATGTA

HVP31
31_fus_3578_
CAGCTGCATGCACAAACCA
617
TCTTTGCTCGCAGCTCGT
618

PVT1_005_

exon1

HVP31
31_unsp_230_231
CGGCATTGGAAATACCCTACGAT
619
TCTTAAACATTTTGTACA
620

CACTCCGTGT

HVP31
31_fus_3578_
CAGCTGCATGCACAAACCA
621
AAATACGGCTGCACCGAGT
622

MYC_001_

exon2

HVP31
31_fus_
AATCGTGTGCCCCAACTGT
623
ATCATGATGGCTGTATGTGCCA
624

877_PVT1_

002_exon3

HVP31
31_fus_877_
AATCGTGTGCCCCAACTGT
625
CTGAGAAGCCCTGCCCTTC
626

MYC_001_exon1

HVP31
31_unsp_877_878
AATCGTGTGCCCCAACTGT
627
CCCCTGTCTGTCTGTCAATTACT
628

G

HVP31
31_sp_877_2646
AATCGTGTGCCCCAACTGT
629
CGTTGAGAAAGAGTCTCCATCG
630

TTTT

HVP31
31_unsp_3590_
CAGCTGCATGCACAAACCA
631
GCCATGTAGATGACACTTGTTCA
632

3591

TACAA

HVP31
31_sp_230_530
CGGCATTGGAAATACCCTACGAT
633
ACATAGTCTTGCAACGTAGGTGT
634

TT

HVP31
31_unsp_412_413
GGAACAACATTAGAAAAATTGAC
635
TTTTCTTCTGGACACAACGGTCT
636

AAACAAAGG

T

HVP31
31_sp_230_2518
CGGCATTGGAAATACCCTACGAT
637
AATGTAAAAACCACCAGTCTGCT
638

ATGTA

HVP31
31_fus_3578_
CAGCTGCATGCACAAACCA
639
ATCATGATGGCTGTATGTGCCA
640

PVT1_002_exon3

HVP31
31_fus_877_
AATCGTGTGCCCCAACTGT
641
TCTTTGCTCGCAGCTCGT
642

PVT1 _005_exon1

HVP31
31_unsp_2517_
CACTAGATGGCAACCCTGTATCT
643
AATGTAAAAACCACCAGTCTGCT
644

2518

ATGTA

HVP31
31_sp_877_3295
AATCGTGTGCCCCAACTGT
645
GAATTCGATGTGGTGGTGTTGTT
646

G

HVP31
31_fus_877_
AATCGTGTGCCCCAACTGT
647
GGTGATCCAGACTCTGACCTTTT
648

MYC_001_exon3

G

HVP31
31_unsp_3294_
CATGCGGGTGGTCAGGTAA
649
GAATTCGATGTGGTGGTGTTGTT
650

3295

G

HVP31
31_fus_877_
AATCGTGTGCCCCAACTGT
651
AAATACGGCTGCACCGAGT
652

MYC_001_exon2

HVP31
31_unsp_529_530
GAAACGATTCCACAACATAGGAGGA
653
ACATAGTCTTGCAACGTAGGTGT
654

TT

HVP31
31_sp_1296_3295
GCGGGTATGGCAATACTGAAGT
655
GAATTCGATGTGGTGGTGTTGTT
656

G

HVP31
31_fus_3578_
CAGCTGCATGCACAAACCA
657
CTGAGAAGCCCTGCCCTTC
658

MYC_001_exon1

HVP31
31_fus_2807_
CAACGTTTAAATGTGTGTCAGGA
659
TCTTTGCTCGCAGCTCGT
660

PVT1_005_exon1
CAAA

HVP31
31_gen_7233_7912
TGTGTGTGTTGTGTATGTTGTCCTT
661
CAACTTTTACTATGGCGTGACAC
662

CTA

HVP31
31_gen_5802_7226
GCTTAGTTTGGGCCTGTGTT
663
ACCACCGGCATATCTATTAGAGT
664

TTTC

HVP31
31_gen_3840_4137
GCATTGTGCTATGCTTTTTGCTTTG
665
ACAACGTAATGGAGAGGTTGCA
666

ATA

HVP31
31_gen_1546_2268
GTGAAACACCAGAATGGATAGAAAGA
667
TGCACATGCATTACTATCACTGT
668

C

CA

HVP33
33_fus_3577_
ACGTACTGCAACTAACTGCACAA
669
GGTGATCCAGACTCTGACCTTTT
670

MYC_001_exon3

G

HVP33
33_fus_3577_
ACGTACTGCAACTAACTGCACAA
671
CATGGTTCCACCAGCGTTATT
672

PVT1_004_exon1

HVP33
33_sp_1316_3351
GATGAGCTAGAAGACAGCGGATATG
673
GTGGTGGTCGGTTATCGTTGT
674

HVP33
33_unsp_894_895
GTGCCCTACCTGTGCACAA
675
TTCTTCTCTCTATGACTGCT
676

TCTACCT

HVP33
33_unsp_2574_2575
TGTGAAACATAGGGCATTAGTGC
677
CATACACTGGGTTACCATTTTCA
678

AATTA

TCAAA

HVP33
33_fus_894_
GTGCCCTACCTGTGCACAA
679
ATCATGATGGCTGTATGTGCCA
680

PVT1_002_exon3

HVP33
33_fus_894_
GTGCCCTACCTGTGCACAA
681
CTGAGAAGCCCTGCCCTTC
682

MYC_001_exon1

HVP33
33_sp_3589_5594
ACGTACTGCAACTAACTGCACAA
683
ATCAGTGCTGACAACTTTAGATA
684

CAGG

HVP33
33_fus_3577_
ACGTACTGCAACTAACTGCACAA
685
TCTTTGCTCGCAGCTCGT
686

PVT1_005_exon1

HVP33
33_sp_231_531
AGCATTGGAGACAACTATACACA
687
CATATTCCTTTAACGTTGGCTTG
688

ACATT

TGT

HVP33
33_unsp_413_414
ATTCTGTATATGGAAATACATTA
689
TCGTTTGTTTAAATCCACATGTC
690

GAACAAACAG

GTTTT

HVP33
33_fus_3577_
ACGTACTGCAACTAACTGCACAA
691
CTGAGAAGCCCTGCCCTTC
692

MYC_001_exon1

HVP33
33_fus_3577_
ACGTACTGCAACTAACTGCACAA
693
AAATACGGCTGCACCGAGT
694

MYC_001_exon2

HVP33
33_sp_231_414
AGCATTGGAGACAACTATACACAA
695
TCGTTTGTTTAAATCCACATGTC
696

CATT

GTTTT

HVP33
33_fus_2863_
GTGCAGGAGAAAATACTAGATCTT
697
CTGAGAAGCCCTGCCCTTC
698

MYC_001_exon1
TACGA

HVP33
33_unsp_1316_1317
GATGAGCTAGAAGACAGCGGATATG
699
CATCCCCCACCCCACTAGAT
700

HVP33
33_fus_3577_
ACGTACTGCAACTAACTGCACAA
701
ATCATGATGGCTGTATGTGCCA
702

PVT1_002_exon3

HVP33
33_fus_2863_
GTGCAGGAGAAAATACTAGATCTT
703
ATCATGATGGCTGTATGTGCCA
704

PVT1_002_exon3
TACGA

HVP33
33_unsp_231_232
AGCATTGGAGACAACTATACACA
705
CGCAAACACAGTTTACATATTCC
706

ACATT

AAATG

HVP33
33_fus_894_
GTGCCCTACCTGTGCACAA
707
TCTTTGCTCGCAGCTCGT
708

PVT1_005_exon1

HVP33
33_fus_894_
GTGCCCTACCTGTGCACAA
709
AAATACGGCTGCACCGAGT
710

MYC_001_exon2

HVP33
33_fus_2863_
GTGCAGGAGAAAATACTAGAT
711
GGTGATCCAGACTCTGACCTTTT
712

MYC_001_exon3
CTTTACGA

G

HVP33
33_sp_231_2575
AGCATTGGAGACAACTATACAC
713
CATACACTGGGTTACCATTTTCA
714

AACATT

TCAAA

HVP33
33_fus_2863_
GTGCAGGAGAAAATACTAGATC
715
CATGGTTCCACCAGCGTTATT
716

PVT1_004_
TTTACGA

exon1

HVP33
33_sp_
GTGCCCTACCTGTGCACAA
717
TGATATTTCCTCCATGGTT
718

894_2702

TTCCTTGTC

HVP33
33_sp_
AGCATTGGAGACAACTATACACA
719
GTGGTGGTCGGTTATCGTTGT
720

231_3351
ACATT

HVP33
33_sp_1316_
GATGAGCTAGAAGACAGCGGATATG
721
CATACACTGGGTTACCATTTTCA
722

2575

TCAAA

HVP33
33_fus_894_
GTGCCCTACCTGTGCACAA
723
GGTGATCCAGACTCTGACCTTTT
724

MYC_001_

G

exon3

HVP33
33_fus_2863_
GTGCAGGAGAAAATACTAGATCTT
725
TCTTTGCTCGCAGCTCGT
726

PVT1_005_exon1
TACGA

HVP33
33_sp_894_3351
GTGCCCTACCTGTGCACAA
727
GTGGTGGTCGGTTATCGTTGT
728

HVP33
33_fus_894_
GTGCCCTACCTGTGCACAA
729
CATGGTTCCACCAGCGTTATT
730

PVT1_004_exon1

HVP33
33_fus_2863_
GTGCAGGAGAAAATACTAGATCTT
731
AAATACGGCTGCACCGAGT
732

MYC_001_exon2
TACGA

HVP33
33_sp_1316_2702
GATGAGCTAGAAGACAGCGGATATG
733
TGATATTTCCTCCATGGTTT
734

TCCTTGTC

HVP33
33_unsp_3350_3351
GGATGCTGCAAAGTATTCTAAAAC
735
GTGGTGGTCGGTTATCGTTGT
736

ACAA

HVP33
33_unsp_
CGATTTCATAATATTTCGGGTCG
737
CATATTCCTTTAACGTTGGCTTG
738

530_531
TTGG

TGT

HVP33
33_sp_
GTGCCCTACCTGTGCACAA
739
ATCAGTGCTGACAACTTTAGATA
740

894_5594

CAGG

HVP33
33_unsp_
ACGTACTGCAACTAACTGCACAA
741
GCCAGGTGGATGACATAGAACT
742

3589_3590

ATACA

HVP33
33_unsp_
TTGTTGTAGACGGTGCTGACTTT
743
ATCAGTGCTGACAACTTTAGATA
744

5593_5594

CAGG

HVP33
33_sp_
GTGCCCTACCTGTGCACAA
745
CATACACTGGGTTACCATTTTCA
746

894_2575

TCAAA

HVP33
33_sp_
AGCATTGGAGACAACTATACACAA
747
TGATATTTCCTCCAT
748

231_2702
CATT

GGTTTTCCTTGTC

HVP33
33_gen_
CCATTTCTACCTATGCTTGGTTGCT
749
GTTGTGTCATATGCTGTGCATGA
750

3839_4175

AA

HVP33
33_gen_
CTTGCCCTACCCTGCATTG
751
CGGTTAGGCATACAAAATGGAG
752

7292_7909

GAAAT

HVP33
33_gen_
CGGAGCCAAACATGTGCATTG
753
CGTTATCATATGCCCACTGTACC
754

1566_2325

ATT

HVP33
33_gen_
CATGTGTAGGCCTTGAAATAGGTAGAG
755
CCTATTATCAGCACCCGGTTGT
756

5844_7285

HVP35
35_unsp_
CGAGGTAGAAGAAAGCATCCATGAAAT
757
CATACTCCATATGGCTGGCCTTC
758

232_233

HVP35
35_unsp_
TCTACATCTGACTGCACAAACAAAGA
759
CCATCTCCATGTAGATGAAGCAT
760

3596_3597

CTTG

HVP35
35_sp_
CGAGGTAGAAGAAAGCATCCATGAAAT
761
GGAAAGCGTCTCCATCATTTTCT
762

232_2670

TTG

HVP35
35_fus_
ATTACGAGACTGATAGCACATGTTTGT
763
GGTGATCCAGACTCTGACCTTTT
764

2831_MYC_

G

001_exon3

HVP35
35_fus_
TCTACATCTGACTGCACAAACAAAGA
765
GGTGATCCAGACTCTGACCTTTT
766

3584_MYC_

G

001_exon3

HVP35
35_fus_
CGGCTGTTCACAGAGAGCATAAT
767
AAATACGGCTGCACCGAGT
768

883_MYC_

001_exon2

HVP35
35_fus_
ATTACGAGACTGATAGCACATGTTTGT
769
CATGGTTCCACCAGCGTTATT
770

2831_PVT1_

004_exon1

HVP35
35_unsp_
GGGTGAUTTTATTTACACCCTAGTT
771
CATCAGTGCTAACAACCTTAGAC
772

5600_5601

ACT

HVP35
35_fus_2831_
ATTACGAGACTGATAGCACATGTTTGT
773
TCTTTGCTCGCAGCTCGT
774

PVT1_005_exon1

HVP35
35_sp_3596_5601
TCTACATCTGACTGCACAAACAAAGA
775
CATCAGTGCTAACAACCTTAGAC
776

ACT

HVP35
35_sp_883_2670
CGGCTGTTCACAGAGAGCATAAT
777
GGAAAGCGTCTCCATCATTTTCT
778

TTG

HVP35
35_unsp_883_884
CGGCTGTTCACAGAGAGCATAAT
779
CCCGTACGTCTACTAACTACTGC
780

TT

HVP35
35_fus_2831_
ATTACGAGACTGATAGCACATGTTTGT
781
ATCATGATGGCTGTATGTGCCA
782

PVT1_002_exon3

HVP35
35_fus_883_MYC_
CGGCTGTTCACAGAGAGCATAAT
783
GGTGATCCAGACTCTGACCTTTT
784

001_exon3

G

HVP35
35_sp_883_5601
CGGCTGTTCACAGAGAGCATAAT
785
CATCAGTGCTAACAACCTTAGAC
786

ACT

HVP35
35_fus_883_MYC_
CGGCTGTTCACAGAGAGCATAAT
787
CTGAGAAGCCCTGCCCTTC
788

001_exon1

HVP35
35_fus_3584_
TCTACATCTGACTGCACAAACAAAGA
789
TCTTTGCTCGCAGCTCGT
790

PVT1_005_exon1

HVP35
35_fus_2831_
ATTACGAGACTGATAGCACATGTTTGT
791
AAATACGGCTGCACCGAGT
792

MYC_001_exon2

HVP35
35_sp_232_2543
CGAGGTAGAAGAAAGCATCCATGAAAT
793
TCATTGTGAAATGTAAAGACCAC
794

TACCC

HVP35
35_fus_2831_
ATTACGAGACTGATAGCACATGTTTGT
795
CTGAGAAGCCCTGCCCTTC
796

MYC_001_exon1

HVP35
35_unsp_5766_5767
CATCTACTATCATGCAGGCAGTTCT
797
ACTCTGTATTGCAAACCAGATAC
798

CTTG

HVP35
35_unsp_2669_2670
GGAAACCCAGTGTATGGGCTTAAT
799
GGAAAGCGTCTCCATCATTTTCT
800

TTG

HVP35
35_fus_883_
CGGCTGTTCACAGAGAGCATAAT
801
ATCATGATGGCTGTATGTGCCA
802

PVT1_002_exon3

HVP35
35_fus_3584_
TCTACATCTGACTGCACAAACAAAGA
803
CATGGTTCCACCAGCGTTATT
804

PVT1_004_exon1

HVP35
35_fus_3584_
TCTACATCTGACTGCACAAACAAAGA
805
ATCATGATGGCTGTATGTGCCA
806

PVT1_002_exon3

HVP35
35_fus_883_
CGGCTGTTCACAGAGAGCATAAT
807
TCTTTGCTCGCAGCTCGT
808

PVT1_005_exon1

HVP35
35_fus_3584_
TCTACATCTGACTGCACAAACAAAGA
809
CTGAGAAGCCCTGCCCTTC
810

MYC_001_exon1

HVP35
35_sp_883_3319
CGGCTGTTCACAGAGAGCATAAT
811
GCTTTGGTATGGGTCTCGGT
812

HVP35
35_sp_1305_3319
ATTATTTGAACTACCAGACAGCGGTT
813
GCTTTGGTATGGGTCTCGGT
814

HVP35
35_sp_883_2543
CGGCTGTTCACAGAGAGCATAAT
815
TCATTGTGAAATGTAAAGACCAC
816

TACCC

HVP35
35__sp_1305_2670
ATTATTTGAACTACCAGACAGCGGTT
817
GGAAAGCGTCTCCATCATTTTCT
818

TTG

HVP35
35__sp_232_415
CGAGGTAGAAGAAAGCATCCATGAAAT
819
TCCACCGATGTTATGGAATCGTT
820

TT

HVP35
35_fus_3584_
TCTACATCTGACTGCACAAACAAAGA
821
AAATACGGCTGCACCGAGT
822

MYC_001_exon2

HVP35
35_fus_883_
CGGCTGTTCACAGAGAGCATAAT
823
CATGGTTCCACCAGCGTTATT
824

PVT1_004_exon1

HVP35
35_unsp_414_415
GGAGAAACGTTAGAAAAACAATGCAAC
825
TCCACCGATGTTATGGAATCGTT
826

A

TT

HVP35
35_sp_232_3319
CGAGGTAGAAGAAAGCATCCATGAAAT
827
GCTTTGGTATGGGTCTCGGT
828

HVP35
35_unsp_1305_1306
ATTATTTGAACTACCAGACAGCGGTT
829
GCTACTAGAGGTTATACTATCCC
830

CACT

HVP35
35_sp_883_5767
CGGCTGTTCACAGAGAGCATAAT
831
ACTCTGTATTGCAAACCAGATAC
832

CTTG

HVP35
35_sp_1305_2543
ATTATTTGAACTACCAGACAGCGGTT
833
TCATTGTGAAATGTAAAGACCAC
834

TACCC

HVP35
35_unsp_2542_2543
CATTAGTGCAATTAAAATGCCCACCTT
835
TCATTGTGAAATGTAAAGACCAC
836

TACCC

HVP35
35_sp_3596_5767
TCTACATCTGACTGCACAAACAAAGA
837
ACTCTGTATTGCAAACCAGATAC
838

CTTG

HVP35
35_unsp_3318_3319
AAAATATATGGGAAGTGCATGTGGGT
839
GCTTTGGTATGGGTCTCGGT
840

HVP35
35_gen_3846_4185
CGTTCGCTATTGCTATCTGTGTCATTA
841
GCCAAATATTGTGCATGAGCGTT
842

AATC

HVP35
35_gen_7293_7879
AACATTCCTACCTCAGCAGAACAC
843
TGGGTGGACCACAAGTATGAAA
844

A

HVP35
35_gen_6017_7286
GGTACAGATAACAGGGAATGCATTTCT
845
GACATTCICC1GCI111ACCIGG
846

TTA

HVP35
35_gen_1555_2293
GCTATGTATTTCAGCTGCAAGTATGCT
847
CATTCTGGTGTTTCTCCATCAAC
848

CT

HVP39
39_sp_943_2636
CGTGGTGTGCAACTGCAA
849
CTGTTTTGGTCAAATGGAAATGC
850

ATTAG

HVP39
39_unsp_5642_5643
GCAATAACCATTCAGGGTTCCAATT
851
AGTATTGACAACCTTCGCCACA
852

HVP39
39_unsp_1368_1369
GGTGTATTCCGTGCCAGACA
853
GTACACTGCCGCCATGTTC
854

HVP39
39_sp_1368_3424
GGTGTATTCCGTGCCAGACA
855
GGTCGCGGTGGTGTTTGATAA
856

HVP39
39_sp_235_2636
CACCACCTTGCAGGACATTACAATA
857
CTGTTTTGGTCAAATGGAAATGC
858

ATTAG

HVP39
39_fus_3677_
CACAGTAACAGTACAGGCCACA
859
CATGGTTCCACCAGCGTTATT
860

PVT1_004_exon1

HVP39
39_fus_3677_
CACAGTAACAGTACAGGCCACA
861
ATCATGATGGCTGTATGTGCCA
862

PVT1_002_exon3

HVP39
39_unsp_801_802
CATGCAGTTAATCACCAACATCAACT
863
TGCTGTAGTTGTCGCAGAGTATC
864

HVP39
39_sp_235_418
CACCACCTTGCAGGACATTACAATA
865
CTGTCCTGTATAGCTTCCTGCTA
866

TTTT

HVP39
39_unsp_943_944
CGTGGTGTGCAACTGCAA
867
CACTGTGTCGCCTGTTTGTTTAT
868

HVP39
39_fus_943_MYC_
CGTGGTGTGCAACTGCAA
869
AAATACGGCTGCACCGAGT
870

001_exon2

HVP39
39_unsp_2635_
ATTAGATGGGTATGCAATAAGTTTAGA
871
CTGTTTTGGTCAAATGGAAATGC
872

2636
TAGG

ATTAG

HVP39
39_fus_2927_
ACAACGTTTAAATGTGTTACAGGACA
873
AAATACGGCTGCACCGAGT
874

MYC_001_exon2

HVP39
39_fus_2927_
ACAACGTTTAAATGTGTTACAGGACA
875
ATCATGATGGCTGTATGTGCCA
876

PVT1_002_exon3

HVP39
39_fus_943_
CGTGGTGTGCAACTGCAA
877
ATCATGATGGCTGTATGTGCCA
878

PVT1_002_exon3

HVP39
39_fus_943_
CGTGGTGTGCAACTGCAA
879
TCTTTGCTCGCAGCTCGT
880

PVT1_005_exon1

HVP39
39_fus_943_
CGTGGTGTGCAACTGCAA
881
GGTGATCCAGACTCTGACCTTTT
882

MYC_001_exon3

G

HVP39
39_fus_2927_
ACAACGTTTAAATGTGTTACAGGACA
883
TCTTTGCTCGCAGCTCGT
884

PVT1_005_exon1

HVP39
39_fus_943_
CGTGGTGTGCAACTGCAA
885
CTGAGAAGCCCTGCCCTTC
886

MYC_001_exon1

HVP39
39_sp_943_5643
CGTGGTGTGCAACTGCAA
887
AGTATTGACAACCTTCGCCACA
888

HVP39
39_unsp_3689_
CACAGTAACAGTACAGGCCACA
889
CGTATCCAATGCCAGGTACATG
890

3690

AAA

HVP39
39_sp_3689_5643
CACAGTAACAGTACAGGCCACA
891
AGTATTGACAACCTTCGCCACA
892

HVP39
39_fus_3677_
CACAGTAACAGTACAGGCCACA
893
TCTTTGCTCGCAGCTCGT
894

PVT1_005_exon1

HVP39
39_unsp_417_418
CTCGGACTCGGTGTATGCAA
895
CTGTCCTGTATAGCTTCCTGCTA
896

TTTT

HVP39
39_fus_943_
CGTGGTGTGCAACTGCAA
897
CATGGTTCCACCAGCGTTATT
898

PVT1_004_exon1

HVP39
39_sp_943_3424
CGTGGTGTGCAACTGCAA
899
GGTCGCGGTGGTGTTTGATAA
900

HVP39
39_unsp_235_236
CACCACCTTGCAGGACATTACAATA
901
GATTGGCATGCAGCTAGTGG
902

HVP39
39_sp_235_802
CACCACCTTGCAGGACATTACAATA
903
TGCTGTAGTTGTCGCAGAGTATC
904

HVP39
39_fus_2927_
ACAACGTTTAAATGTGTTACAGGACA
905
GGTGATCCAGACTCTGACCTTTT
906

MYC_001_exon3

G

HVP39
39_fus_2927_
ACAACGTTTAAATGTGTTACAGGACA
907
CTGAGAAGCCCTGCCCTTC
908

MYC_001_exon1

HVP39
39_fus_3677_
CACAGTAACAGTACAGGCCACA
909
AAATACGGCTGCACCGAGT
910

MYC_001_exon2

HVP39
39_sp_1368_2636
GGTGTATTCCGTGCCAGACA
911
CTGTTTTGGTCAAATGGAAATGC
912

ATTAG

HVP39
39_fus_3677_
CACAGTAACAGTACAGGCCACA
913
CTGAGAAGCCCTGCCCTTC
914

MYC_001_exon1

HVP39
39_fus_2927_
ACAACGTTTAAATGTGTTACAGGACA
915
CATGGTTCCACCAGCGTTATT
916

PVT1_004_exon1

HVP39
39_fus_3677_
CACAGTAACAGTACAGGCCACA
917
GGTGATCCAGACTCTGACCTTTT
918

MYC_001_exon3

G

HVP39
39_sp_235_3424
CACCACCTTGCAGGACATTACAATA
919
GGTCGCGGTGGTGTTTGATAA
920

HVP39
39_gen_3939_
TTGGTGTGGTTTGGTGTGTGTATAT
921
CTCCAATGGTGTGGTACGTATAA
922

4242

GAA

HVP39
39_gen_7267_7833
CATTTTGTGGCGACCGAAGT
923
CCTGGACAGGATGATGAGTAAT
924

AAGG

HVP39
39_gen_1618_2386
AGGGTTACTGTAGGAAAGGGATTAAGT
925
CGTATCCCCTGTTACCACACTAA
926

TATTG

HVP39
39_gen_5893_7260
CCAGCCATTGGGTGTTGGTA
927
GCCTATAATGCACAACTGTGTCT
928

GTT

HVP45
45_fus_929_
AGCACCTTGTCCTTTGTGTGT
929
GGTGATCCAGACTCTGACCTTTT
930

MYC_001_exon3

G

HVP45
45_fus_2901_
CGTTACAGGACAAAATACTAGACCACT
931
CATGGTTCCACCAGCGTTATT
932

PVT1_004_exon1
A

HVP45
45_sp_929_2610
AGCACCTTGTCCTTTGTGTGT
933
GAAATGCATGTGGAAATGTAAA
934

TACCGT

HVP45
45_fus_3648V_
TCCTGTGTTCAAGTACAAGTAACAACA
935
AAATACGGCTGCACCGAGT
936

MYC_001_exon2
A

HVP45
45_sp_1357_2610
TCAGATAGTGGCTATGGCTGTTCT
937
GAAATGCATGTGGAAATGTAAA
938

TACCGT

HVP45
45_fus_3648_
TCCTGTGTTCAAGTACAAGTAACAACA
939
GGTGATCCAGACTCTGACCTTTT
940

MYC_001_exon3
A

G

HVP45
45_sp_230_791
CTACAAGACGTATCTATTGCCTGTGT
941
TCAAAAACAGCTGCTGTAGTGTT
942

CT

HVP45
45_sp_929_3423
AGCACCTTGTCCTTTGTGTGT
943
CCCACGGATGCGGTTTTG
944

HVP45
45_unsp_412_413
AAACTCTGTATATGGAGAGACACTGGA
945
CGTTTGTCCTTAAGGTGTCTACG
946

TTTT

HVP45
45_sp_230_413
CTACAAGACGTATCTATTGCCTGTGT
947
CGTTTGTCCTTAAGGTGTCTACG
948

TTTT

HVP45
45_sp_230_2737
CTACAAGACGTATCTATTGCCTGTGT
949
GGATTCCTTCGGTGTCTGCAT
950

HVP45
45_fus_2901_
CGTTACAGGACAAAATACTAGACCACT
951
CTGAGAAGCCCTGCCCTTC
952

MYC_001_exon1
A

HVP45
45_unsp_230_231
CTACAAGACGTATCTATTGCCTGTGT
953
AAGTCTATACATTTATGGCATGC
954

AGCATA

HVP45
45_fus_929_
AGCACCTTGTCCTTTGTGTGT
955
CATGGTTCCACCAGCGTTATT
956

PVT1_004_exon1

HVP45
45_sp_1357_3423
TCAGATAGTGGCTATGGCTGTTCT
957
CCCACGGATGCGGTTTTG
958

HVP45
45_fus_929_
AGCACCTTGTCCTTTGTGTGT
959
AAATACGGCTGCACCGAGT
960

MYC_001_exon2

HVP45
45_fus_929_
AGCACCTTGTCCTTTGTGTGT
961
TCTTTGCTCGCAGCTCGT
962

PVT1_005_exon1

HVP45
45_sp_230_3423
CTACAAGACGTATCTATTGCCTGTGT
963
CCCACGGATGCGGTTTTG
964

HVP45
45_unsp_2609_2610
CATTATTACAGCTAAAATGTCCT
965
GAAATGCATGTGGAAATGTAAA
966

CCAATCC

TACCGT

HVP45
45_fus_3648_
TCCTGTGTTCAAGTACAAGTAACAACA
967
ATCATGATGGCTGTATGTGCCA
968

PVT1_002_exon3
A

HVP45
45_fus_2901_
CGTTACAGGACAAAATACTAGACCACT
969
ATCATGATGGCTGTATGTGCCA
970

PVT1_002_exon3
A

HVP45
45_fus_2901_
CGTTACAGGACAAAATACTAGACCACT
971
AAATACGGCTGCACCGAGT
972

MYC_001_exon2
A

HVP45
45_unsp_790_791
GGAGTTAGTCATGCACAACTACCA
973
TCAAAAACAGCTGCTGTAGTGTT
974

CT

HVP45
45_sp_929_2737
AGCACCTTGTCCTTTGTGTGT
975
GGATTCCTTCGGTGTCTGCAT
976

HVP45
45_fus_3648_
TCCTGTGTTCAAGTACAAGTAACAACA
977
TCTTTGCTCGCAGCTCGT
978

PVT1_005_exon1
A

HVP45
45_sp_929_5608
AGCACCTTGTCCTTTGTGTGT
979
GCTGACAACTCTGGCCACA
980

HVP45
45_sp_230_2610
CTACAAGACGTATCTATTGCCTGTGT
981
GAAATGCATGTGGAAATGTAAA
982

TACCGT

HVP45
45_unsp_5607_5608
GCACACAATATTATTTATGGCCATGGTA
983
GCTGACAACTCTGGCCACA
984

HVP45
45_fus_2901_
CGTTACAGGACAAAATACTAGACCACT
985
TCTTTGCTCGCAGCTCGT
986

PVT1_005_exon1
A

HVP45
45_unsp_929_930
AGCACCTTGTCCTTTGTGTGT
987
CAATTGTTTCTACAAAGAACCAG
988

CCATT

HVP45
45_fus_2901_
CGTTACAGGACAAAATACTAGACCACT
989
GGTGATCCAGACTCTGACCTTTT
990

MYC_001_exon3
A

G

HVP45
45_fus_929_
AGCACCTTGTCCTTTGTGTGT
991
CTGAGAAGCCCTGCCCTTC
992

MYC_001_exon1

HVP45
45_sp_1357_2737
TCAGATAGTGGCTATGGCTGTTCT
993
GGATTCCTTCGGTGTCTGCAT
994

HVP45
45_unsp_1357_1358
TCAGATAGTGGCTATGGCTGTTCT
995
ACTATCCCCACCACTACTTTGTG
996

TA

HVP45
45_fus_3648_
TCCTGTGTTCAAGTACAAGTAACAACA
997
CATGGTTCCACCAGCGTTATT
998

PVT1_004_exon1
A

HVP45
45_sp_3660_5608
TCCTGTGTTCAAGTACAAGTAACAACA
999
GCTGACAACTCTGGCCACA
1000

A

HVP45
45_fus_3648_
TCCTGTGTTCAAGTACAAGTAACAACA
1001
CTGAGAAGCCCTGCCCTTC
1002

MYC_001_exon1
A

HVP45
45_fus_929_
AGCACCTTGTCCTTTGTGTGT
1003
ATCATGATGGCTGTATGTGCCA
1004

PVT1_002_exon3

HVP45
45_gen_3910_4227
TGCTTTTGCTTGGTTGTTGGT
1005
CATCACAGGTATGTTACACTGTA
1006

CTGT

HVP45
45_unsp_3660_3661
TCCTGTGTTCAAGTACAAGTAACAACA
1007
GGTCTGCATATTTGCGTAGCCTA
1008

A

TA

HVP45
45_gen_7316_7858
ATTTCGGTTGCCTGTGGCTTATA
1009
CAGTTGTGCAAGCCATTGTTTTA
1010

GT

HVP45
45_sp_3750_5608
CGCAAATATGCAGACCATTACTCAGAA
1011
GCTGACAACTCTGGCCACA
1012

HVP45
45_gen_1607_2360
6CAACGTTATAC6CCCATATCCAAT
1013
GGTACGTGCAACAATGTGCTTA
1014

A

HVP45
45_unsp_3750_3751
CGCAAATATGCAGACCATTACTCAGAA
1015
CCCACCGAGATTTGTACACTGTT
1016

A

HVP45
45_gen_5858_7309
GGCATGTGTAGGTATGGAAATTGGT
1017
ACATCCTGCGTAATAACAGCTGT
1018

AG

HVP45
45_unsp_3422_3423
TGACGACACGGTATCCGCTA
1019
CCCACGGATGCGGTTTTG
1020

HVP51
51_unsp_2547_2548
AGTATGTCCACCATTACTAATAACGTCA
1021
TCATTCAATGTATACACAGCATT
1022

AAC

CCCAT

HVP51
51_fus_3572_MYC_
CTAACACTGGAGGGCACCAAA
1023
AAATACGGCTGCACCGAGT
1024

001_exon2

HVP51
51_fus_886_
GGGCGAACTAAGCCTGGTTT
1025
ATCATGATGGCTGTATGTGCCA
1026

PVT1_002_exon3

HVP51
51_fus_886_
GGGCGAACTAAGCCTGGTTT
1027
AAATACGGCTGCACCGAGT
1028

MYC_001_

exon2

HVP51
51_fus_3572_
CTAACACTGGAGGGCACCAAA
1029
TCTTTGCTCGCAGCTCGT
1030

PVT1_005_

exon1

HVP51
51_fus_
GTGCCAGGAGAAAATACTAGACTGTTA
1031
CTGAGAAGCCCTGCCCTTC
1032

2834_MYC_
T

001_exon1

HVP51
51_unsp_
CTAACACTGGAGGGCACCAAA
1033
ATGCCAGGTTGAGGATACGTTTT
1034

3584_3585

TAT

HVP51
51_fus_
GTGCCAGGAGAAAATACTAGACTGTTA
1035
CATGGTTCCACCAGCGTTATT
1036

2834_PVT1_
T

004_exon1

HVP51
51_unsp_
CGGACAGCGGATATGGCAATA
1037
TCTGTTGTTTCCACATCCATAAC
1038

1302_1303

ACT

HVP51
51_fus_
CTAACACTGGAGGGCACCAAA
1039
ATCATGATGGCTGTATGTGCCA
1040

3572_PVT1_

002_exon3

HVP51
51_sp_3584_
CTAACACTGGAGGGCACCAAA
1041
CAATTCGAGACACAGGTGCAG
1042

5521

HVP51
51_unsp_
GAGAGTATAGACGTTATAGCAGGTCTG
1043
TCCCGCTATTTCATGGAACC
1044

401_402
T

TTTT

HVP51
51_sp_
CGGACAGCGGATATGGCAATA
1045
CCACGCAGGTGGTAAGGG
1046

1302_3319

HVP51
51_sp_217_751
CTGCATGAATTATGTGAAGCTTTGAAC
1047
CATCTGCTGTACAACGCGAAG
1048

HVP51
51_fus_886_MYC_
GGGCGAACTAAGCCTGGTTT
1049
GGTGATCCAGACTCTGACCTTTT
1050

001_exon3

G

HVP51
51_fus_3572_
CTAACACTGGAGGGCACCAAA
1051
CATGGTTCCACCAGCGTTATT
1052

PVT1_004_exon1

HVP51
51_sp_1302_2548
CGGACAGCGGATATGGCAATA
1053
TCATTCAATGTATACACAGCATT
1054

CCCAT

HVP51
51_unsp_886_887
GGGCGAACTAAGCCTGGTTT
1055
CTCATCATCCGAAACATTATCTC
1056

CTGT

HVP51
51_fus_2834_
GTGCCAGGAGAAAATACTAGACTGTTA
1057
ATCATGATGGCTGTATGTGCCA
1058

PVT1_002_exon3
T

HVP51
51_unsp_3318_3319
GCACAACAGTGGGAGGTCTATATG
1059
CCACGCAGGTGGTAAGGG
1060

HVP51
51_fus_2834_
GTGCCAGGAGAAAATACTAGACTGTTA
1061
AAATACGGCTGCACCGAGT
1062

MYC_001_exon2
T

HVP51
51_sp_886_5521
GGGCGAACTAAGCCTGGTTT
1063
CAATTCGAGACACAGGTGCAG
1064

HVP51
51_fus_3572_
CTAACACTGGAGGGCACCAAA
1065
GGTGATCCAGACTCTGACCTTTT
1066

MYC_001_exon3

G

HVP51
51_sp_217_402
CTGCATGAATTATGTGAAGCTTTGAAC
1067
TCCCGCTATTTCATGGAACC
1068

TTTT

HVP51
51_unsp_750_751
GCGTGACCAGCTACCAGAAA
1069
CATCTGCTGTACAACGCGAAG
1070

HVP51
51_fus_886_
GGGCGAACTAAGCCTGGTTT
1071
CATGGTTCCACCAGCGTTATT
1072

PVT1_004_exon1

HVP51
51_fus_3572_
CTAACACTGGAGGGCACCAAA
1073
CTGAGAAGCCCTGCCCTTC
1074

MYC_001_exon1

HVP51
51_unsp_217_218
CTGCATGAATTATGTGAAGCTTTGAAC
1075
GTAAACATTGTTTGCATACTGCA
1076

TATGGA

HVPS1
51_fus_886_
GGGCGAACTAAGCCTGGTTT
1077
CTGAGAAGCCCTGCCCTTC
1078

MYC_001_exon1

HVP51
51_sp_886_3319
GGGCGAACTAAGCCTGGTTT
1079
CCACGCAGGTGGTAAGGG
1080

HVPS1
51_fus_2834_
GTGCCAGGAGAAAATACTAGACTGTTA
1081
GGTGATCCAGACTCTGACCTTTT
1082

MYC_001_exon3
T

G

HVP51
51_fus_2834_
GTGCCAGGAGAAAATACTAGACTGTTA
1083
TCTTTGCTCGCAGCTCGT
1084

PVT1_005_exon1
T

HVP51
51_fus_886_
GGGCGAACTAAGCCTGGTTT
1085
TCTTTGCTCGCAGCTCGT
1086

PVT1_005_exon1

HVP51
51_unsp_5520_5521
GGCCCTATACACATTTACTACGCAAA
1087
CAATTCGAGACACAGGTGCAG
1088

HVP51
51_sp_217_3319
CTGCATGAATTATGTGAAGCTTTGAAC
1089
CCACGCAGGTGGTAAGGG
1090

HVP51
51_sp_886_2548
GGGCGAACTAAGCCTGGTTT
1091
TCATTCAATGTATACACAGCATT
1092

CCCAT

HVP51
51_sp_217_2548
CTGCATGAATTATGTGAAGCTTTGAAC
1093
TCATTCAATGTATACACAGCATT
1094

CCCAT

HVP51
51_gen_7175_7808
GGGTATTACATTATCCCCGTAGGTCAA
1095
GCTGCAGCTGTAACAAAATGGA
1096

A

HVP51
51_gen_3834_4103
AAGCCAATATGTGCTGCTAATTGTA
1097
AACACGTATTGGGACAGCAGTA
1098

G

HVPS1
51_gen_1552_2298
GATGGAGGCAACTGGAGAGAAATT
1099
GTGTTTGGTGGGCCATATATGA
1100

CTAT

HVP51
51_gen_5771_7168
ACACCCCTCCACAGGCTAA
1101
TGTACGCCAACCTGCAACAA
1102

HVP52
52_unsp_523_524
GACATGTTAATGCAAACAAGCGATTTC
1103
TCAGTTGTTTCAGGTTGCAGATC
1104

TAATA

HVPS2
52_fus_879_
GCTGTTGGGCACATTACAAGTT
1105
CTGAGAAGCCCTGCCCTTC
1106

MYC_001_exon1

HVP52
52_fus_2857_
GCTGATAGTAATGACCTAAACGCACAA
1107
TCTTTGCTCGCAGCTCGT
1108

PVT1_005_exon1
A

HVP52
52_fus_879_
GCTGTTGGGCACATTACAAGTT
1109
CATGGTTCCACCAGCGTTATT
1110

PVT1_004_exon1

HVP52
52_sp_224_524
AGAATCGGTGCATGAAATAAGGCT
1111
TCAGTTGTTTCAGGTTGCAGATC
1112

TAATA

HVP52
52_sp_3625_5643
TCACTGCAACTGAGTGCACAA
1113
TGCTTACAACCTTAGAGACAGGT
1114

ACA

HVP52
52_fus_3613_
TCACTGCAACTGAGTGCACAA
1115
TCTTTGCTCGCAGCTCGT
1116

PVT1_005_exon1

HVP52
52_unsp_5642_5643
TTTTACTACGTCGCAGGCGTAA
1117
TGCTTACAACCTTAGAGACAGGT
1118

ACA

HVP52
52_sp_224_407
AGAATCGGTGCATGAAATAAGGCT
1119
CGCTTGTTTGCATTAACATGTCT
1120

TTCT

HVP52
52_unsp_3625_3626
TCACTGCAACTGAGTGCACAA
1121
TGCCAGGTAGATGAAATTTGAA
1122

CATACA

HVPS2
52_fus_879_
GCTGTTGGGCACATTACAAGTT
1123
GGTGATCCAGACTCTGACCTTTT
1124

MYC_001_exon3

G

HVP52
52_fus_879_
GCTGTTGGGCACATTACAAGTT
1125
AAATACGGCTGCACCGAGT
1126

MYC_001_exon2

HVP52
52_sp_879_3345
GCTGTTGGGCACATTACAAGTT
1127
GCGGAGGTCTTGGAGGTTT
1128

HVP52
52_fus_3613_
TCACTGCAACTGAGTGCACAA
1129
ATCATGATGGCTGTATGTGCCA
1130

PVT1_002_exon3

HVP52
52_fus_2857_
GCTGATAGTAATGACCTAAACGCACAA
1131
GGTGATCCAGACTCTGACCTTTT
1132

MYC_001_exon3
A

G

HVP52
S2_fus_879_
GCTGTTGGGCACATTACAAGTT
1133
ATCATGATGGCTGTATGTGCCA
1134

PVT1_002_exon3

HVP52
52_sp_224_738
AGAATCGGTGCATGAAATAAGGCT
1135
GCATTTGCTGTAGAGTACGAAG
1136

GT

HVP52
52_sp_224_3345
AGAATCGGTGCATGAAATAAGGCT
1137
GCGGAGGTCTTGGAGGTTT
1138

HVP52
52_sp_1301_2569
CAAACCATGTCACGTAGAAGACAG
1139
GGGTTTTTGAAATGAAACACAA
1140

CCAATC

HVPS2
52_unsp_737_738
GATGAGGAGGATACAGATGGTGTG
1141
GCATTTGCTGTAGAGTACGAAG
1142

GT

HVP52
52_unsp_1301_1302
CAAACCATGTCACGTAGAAGACAG
1143
CCCCACCCCACTTGATTGA
1144

HVP52
52_sp_1301_2696
CAAACCATGTCACGTAGAAGACAG
1145
CGGTATCGACTCCATCGTTTTCC
1146

HVP52
52_fus_28S7_
GCTGATAGTAATGACCTAAACGCACAA
1147
CTGAGAAGCCCTGCCCTTC
1148

MYC_001_exon1
A

HVP52
52_sp_879_S810
GCTGTTGGGCACATTACAAGTT
1149
CCTGTATTGCAGGCCAGACA
1150

HVP52
52_unsp_224_225
AGAATCGGTGCATGAAATAAGGCT
1151
CACACGCCATATGGATTATTGTC
1152

TCTA

HVP52
52_sp_879_2696
GCTGTTGGGCACATTACAAGTT
1153
CGGTATCGACTCCATCGTTTTCC
1154

HVP52
52_fus_3613_
TCACTGCAACTGAGTGCACAA
1155
GGTGATCCAGACTCTGACCTTTT
1156

MYC_001_exon3

G

HVP52
52_unsp_879_880
GCTGTTGGGCACATTACAAGTT
1157
TCCTCTGAAATGTTATCTCCTG
1158

TTTGTT

HVP52
52_unsp_2568_2569
CCTTAGTACAAATAAAATGCCCACCAT
1159
GGGTTTTTGAAATGAAACACAA
1160

CCAATC

HVP52
52_fus_2857_
GCTGATAGTAATGACCTAAACGCACAA
1161
ATCATGATGGCTGTATGTGCCA
1162

PVT1_002_exon3
A

HVP52
52_sp_224_2696
AGAATCGGTGCATGAAATAAGGCT
1163
CGGTATCGACTCCATCGTTTTCC
1164

HVP52
52_fus_28S7_
GCTGATAGTAATGACCTAAACGCACAA
1165
AAATACGGCTGCACCGAGT
1166

MYC_001_exon2
A

HVP52
52_fus_879_
GCTGTTGGGCACATTACAAGTT
1167
TCTTTGCTCGCAGCTCGT
1168

PVT1_005_exon1

HVP52
52_unsp_3344_3345
GTAACAGGAGTATGGGAAGTACATGT
1169
GCGGAGGTCTTGGAGGTTT
1170

G

HVP52
52_unsp_406_407
GTATGGGAAAACATTAGAAGAGAGGG
1171
CGCTTGTTTGCATTAACATGTCT
1172

T

TTCT

HVP52
52_sp_1301_3345
CAAACCATGTCACGTAGAAGACAG
1173
GCGGAGGTCTTGGAGGTTT
1174

HVP52
52_fus_3613_
TCACTGCAACTGAGTGCACAA
1175
AAATACGGCTGCACCGAGT
1176

MYC_001_exon2

HVP52
52_sp_879_2569
GCTGTTGGGCACATTACAAGTT
1177
GGGTTTTTGAAATGAAACACAA
1178

CCAATC

HVP52
52_sp_224_2569
AGAATCGGTGCATGAAATAAGGCT
1179
GGGTTTTTGAAATGAAACACAA
1180

CCAATC

HVP52
52_fus_3613_
TCACTGCAACTGAGTGCACAA
1181
CATGGTTCCACCAGCGTTATT
1182

PVT1_004_exon1

HVP52
S2_sp_3625_5810
TCACTGCAACTGAGTGCACAA
1183
CCTGTATTGCAGGCCAGACA
1184

HVP52
52_sp_879_5643
GCTGTTGGGCACATTACAAGTT
1185
TGCTTACAACCTTAGAGACAGGT
1186

ACA

HVP52
52_unsp_5809_5810
AAGCATCTATTATTATGCAGGCAGTTCT
1187
CCTGTATTGCAGGCCAGACA
1188

HVP52
52_fus_3613_
TCACTGCAACTGAGTGCACAA
1189
CTGAGAAGCCCTGCCCTTC
1190

MYC_001_exon1

HVP52
52_fus_2857_
GCTGATAGTAATGACCTAAACGCACAA
1191
CATGGTTCCACCAGCGTTATT
1192

PVT1_004_exon1
A

HVP52
52_gen_6060_7338
GGACTATATGTTTTGGGAGGTGGATTT
1193
GATGCAGGGCGTTTTAGTTTGG
1194

HVP52
52_gen_1551_2319
CACCATCAGTTGCAGAAGGATTAAAAG
1195
CTGTGACATTAGTTTGGACACTG
1196

TT

HVP52
52_gen_7345_7942
TCGGTTGGTCTTGGCACAA
1197
TTTAGGCGGGACAACAAGTGT
1198

HVP52
52_gen_3875_4225
CAACACAAGCCAATATTGCTGCTA
1199
CCTGCGCATACACCGATATAGAT
1200

HVP56
56_unsp_910_911
GTTAACAGTAACGTGCCCACTCT
1201
TTCTACAATTGCCTCTACTTC
1202

AAACCAT

HVP56
56_fus_3629_
ACAACAACCACCCTGGTGATAAG
1203
AAATACGGCTGCACCGAGT
1204

MYC_001_exon2

HVP56
56_fus_2861_
GTGCCAGAACAAAATACTAGACTGTTT
1205
ATCATGATGGCTGTATGTGCCA
1206

PVT1_002_exon3

HVP56
S6_fus_910_
GTTAACAGTAACGTGCCCACTCT
1207
GGTGATCCAGACTCTGACCTTTT
1208

MYC_001_exon3

G

HVP56
56_sp_910_2572
GTTAACAGTAACGTGCCCACTCT
1209
TGAAACTGAAACACTAACATTCT
1210

ACTGTGT

HVP56
56_fus_2861_
GTGCCAGAACAAAATACTAGACTGTTT
1211
TCTTTGCTCGCAGCTCGT
1212

PVT1_005_exon1

HVPS6
S6_fus_2861_
GTGCCAGAACAAAATACTAGACTGTTT
1213
CATGGTTCCACCAGCGTTATT
1214

PVT1_004_exon1

HVP56
56_fus_3629_
ACAACAACCACCCTGGTGATAAG
1215
CTGAGAAGCCCTGCCCTTC
1216

MYC_001_exon1

HVP56
56_unsp_3641_3642
ACAACAACCACCCTGGTGATAAG
1217
TATTGTCTGTACTTGTCCAATGA
1218

TATGT

HVP56
56_unsp_532_533
TGCATTGTGACAGAAAAAGACGATTTC
1219
ACGTCTTGCAGCGTTGGTA
1220

HVP56
56_unsp_26982699
AGAATGTTAGTGTTTCAGTTTCAAAATC
1221
TTTTCTTTGTCCTCGTCGTTA
1222

C

TCCAA

HVP56
56_fus_3629_
ACAACAACCACCCTGGTGATAAG
1223
ATCATGATGGCTGTATGTGCCA
1224

PVT1_002_exon3

HVP56
56_fus_2861_
GTGCCAGAACAAAATACTAGACTGTTT
1225
CTGAGAAGCCCTGCCCTTC
1226

MYC_001_exon1

HVP56
56_sp_233_2572
GCACCACTTGAGTGAGGTATTAGAA
1227
TGAAACTGAAACACTAACATTCT
1228

ACTGTGT

HVP56
56_unsp_772_773
ACAGCAAGCTAGACAAGCTAAACAA
1229
TGTACAACACGCAGGTCCTC
1230

HVP56
56_fus_910_
GTTAACAGTAACGTGCCCACTCT
1231
CTGAGAAGCCCTGCCCTTC
1232

MYC_001_exon1

HVP56
56_sp_910_5597
GTTAACAGTAACGTGCCCACTCT
1233
ACAACCTTTGAAACAGGTGTTG
1234

GA

HVP56
56_sp_233_533
GCACCACTTGAGTGAGGTATTAGAA
1235
ACGTCTTGCAGCGTTGGTA
1236

HVP56
56_fus_2861_
GTGCCAGAACAAAATACTAGACTGTTT
1237
GGTGATCCAGACTCTGACCTTTT
1238

MYC_001_exon3

G

HVP56
56_unsp_5758_5759
ATCATGCAGGCAGTTCACGA
1239
CAACCGTACCCTAAATACCCTAT
1240

ATTGA

HVP56
56_sp_1295_3349
CAAGACAGCGGGTATGGCAATA
1241
GGTGGTGGTGGTGGTCTT
1242

HVP56
56_sp_1295_2699
CAAGACAGCGGGTATGGCAATA
1243
TTTTCTTTGTCCTCGTCGTTAT
1244

CCAA

HVP56
56_unsp_233_234
GCACCACTTGAGTGAGGTATTAGAA
1245
ACAATAAACATACTCTGCACACT
1246

GCATA

HVP56
56_sp_3641_5597
ACAACAACCACCCTGGTGATAAG
1247
ACAACCTTTGAAACAGGTGTTG
1248

GA

HVP56
56_sp_233_2699
GCACCACTTGAGTGAGGTATTAGAA
1249
TTTTCTTTGTCCTCGTCGTTAT
1250

CCAA

HVP56
56_sp_1295_2572
CAAGACAGCGGGTATGGCAATA
1251
TGAAACTGAAACACTAACATTCT
1252

ACTGTGT

HVP56
56_sp_910_3349
GTTAACAGTAACGTGCCCACTCT
1253
GGTGGTGGTGGTGGTCTT
1254

HVP56
56_unsp_1295_1296
CAAGACAGCGGGTATGGCAATA
1255
GGTACTGTTTTGTGAGCCTCCAT
1256

TT

HVP56
56_fus_910_
GTTAACAGTAACGTGCCCACTCT
1257
AAATACGGCTGCACCGAGT
1258

MYC_001_exon2

HVP56
56_fus_3629_
ACAACAACCACCCTGGTGATAAG
1259
GGTGATCCAGACTCTGACCTTTT
1260

MYC_001_exon3

G

HVP56
56_fus_3629_
ACAACAACCACCCTGGTGATAAG
1261
CATGGTTCCACCAGCGTTATT
1262

PVT1_004_exon1

HVP56
56_fus_2861_
GTGCCAGAACAAAATACTAGACTGTTT
1263
AAATACGGCTGCACCGAGT
1264

MYC_001_exon2

HVP56
56_unsp_5596_5S97
AGGGATCCTCCTTTGCATTATGG
1265
ACAACCTTTGAAACAGGTGTTG
1266

GA

HVP56
56_sp_233_416
GCACCACTTGAGTGAGGTATTAGAA
1267
CAATTGCTTTTCCTCCGGAGTTA
1268

A

HVP56
56_sp_910_5759
GTTAACAGTAACGTGCCCACTCT
1269
CAACCGTACCCTAAATACCCTAT
1270

ATTGA

HVP56
56_fus_910_
GTTAACAGTAACGTGCCCACTCT
1271
ATCATGATGGCTGTATGTGCCA
1272

PVT1_002_exon3

HVP56
56_sp_910_2699
GTTAACAGTAACGTGCCCACTCT
1273
TTTTCTTTGTCCTCGTCGTTAT
1274

CCAA

HVP56
56_unsp_415_416
TCAGTGTATGGAGCTACACTAGAAAGT
1275
CAATTGCTTTTCCTCCGGAGTTA
1276

A

HVP56
56_fus_910_
GTTAACAGTAACGTGCCCACTCT
1277
TCTTTGCTCGCAGCTCGT
1278

PVT1_005_exon1

HVP56
56_sp_3641_5759
ACAACAACCACCCTGGTGATAAG
1279
CAACCGTACCCTAAATACCCTAT
1280

ATTGA

HVP56
56_fus_910_
GTTAACAGTAACGTGCCCACTCT
1281
CATGGTTCCACCAGCGTTATT
1282

PVT1_004_exon1

HVP56
56_fus_3629_
ACAACAACCACCCTGGTGATAAG
1283
TCTTTGCTCGCAGCTCGT
1284

PVT1_005_exon1

HVP56
56_sp_233_3349
GCACCACTTGAGTGAGGTATTAGAA
1285
GGTGGTGGTGGTGGTCTT
1286

HVP56
56_sp_233_773
GCACCACTTGAGTGAGGTATTAGAA
1287
TGTACAACACGCAGGTCCTC
1288

HVP56
56_gen_3891_4183
TGCTACGCATATATATTGCAA
1289
GGATGTGGCTATAACAAACCAA
1290

CCATTGA

AACAAT

HVP56
56_gen_7283_7844
AATTCGGTTGCATGGCCTAGT
1291
GGGTGCGGTACTGTACATAATT
1292

CAAG

HVP56
56_gen_6009_7276
TGTACTCCCGCTATGGGTGAA
1293
GTGTCTATCATGTCCCCATCCTCT
1294

A

HVP56
56_gen_1545_2322
CAGATGATAGCCAAATTGCGTTTCA
1295
GCTGTTGTGCCCTTTTATAATGT
1296

CTAC

HVP58
58_sp_898_5643
TGCTTATGGGCACATGTACCATT
1297
GCTTACAACCTTAGACACAGGC
1298

A

HVP58
58_fus_3596_
GAGGAGGACTACACAGTACAACTAACT
1299
ATCATGATGGCTGTATGTGCCA
1300

PVT1_002_exon3

HVP58
58_fus_898_
TGCTTATGGGCACATGTACCATT
1301
GGTGATCCAGACTCTGACCTTTT
1302

MYC_001_exon3

G

HVP58
58_sp_1320_3355
AAAATTATTGAGCTAGAAGACAGCGGA
1303
CCCTGTGTACTTTCGTTGTTGGT
1304

T

HVP58
58_unsp_1320_1321
AAAATTATTGAGCTAGAAGACAGCGGA
1305
CCCCACTAGACTCCGAGTCATTT
1306

T

AA

HVP58
58_fus_3596_
GAGGAGGACTACACAGTACAACTAACT
1307
GGTGATCCAGACTCTGACCTTTT
1308

MYC_001_exon3

G

HVP58
58_fus_2867_
GCAGGACAAAATCCTAGACATATACGA
1309
TCTTTGCTCGCAGCTCGT
1310

PVT1_005_exon1
A

HVP58
58_fus_3596_
GAGGAGGACTACACAGTACAACTAACT
1311
CATGGTTCCACCAGCGTTATT
1312

PVT1_004_exon1

HVP58
58_unsp_898_
TGCTTATGGGCACATGTACCATT
1313
CTGTTCTTCGTTCTATTACCGC
1314

899

CTA

HVP58
58_fus_898_
TGCTTATGGGCACATGTACCATT
1315
ATCATGATGGCTGTATGTGCCA
1316

PVT1_002_

exon3

HVP58
58_unsp_2578_
ATTAGATGGTAACGACATTTCAATAGA
1317
TGCATCAAATGGAAATGGATTG
1318

2579
TGT

TTAAATTCA

HVP58
58_sp_232_2579
GTCAGGCGTTGGAGACATCT
1319
TGCATCAAATGGAAATGGATTG
1320

TTAAATTCA

HVP58
58_fus_2867_
GCAGGACAAAATCCTAGACATATACGA
1321
GGTGATCCAGACTCTGACCTTTT
1322

MYC_001_
A

G

exon3

HVP58
58_fus_898_
TGCTTATGGGCACATGTACCATT
1323
AAATACGGCTGCACCGAGT
1324

MYC_001_exon2

HVP58
58_sp_232_415
GTCAGGCGTTGGAGACATCT
1325
CGACCCGAAATATTATGAAACCT
1326

TTTGT

HVP58
58_fus_898_
TGCTTATGGGCACATGTACCATT
1327
TCTTTGCTCGCAGCTCGT
1328

PVT1_005_

exon1

HVP58
58_sp_232_
GTCAGGCGTTGGAGACATCT
1329
TGATATTTCCTCCATCG
1330

2706

TTTTCCTTGTC

HVP58
58_unsp_
CGCTATATGGAGACACATTAGAACAAA
1331
CGACCCGAAATATTATGAAACCT
1332

414_415
CA

TTTGT

HVP58
58_unsp_
ACAATTATGGGAGGTACATGTGGGTA
1333
CCCTGTGTACTTTCGTTGTTGGT
1334

3354_3355

HVP58
58_sp_1320_
AAAATTATTGAGCTAGAAGACAGCGGA
1335
TGCATCAAATGGAAATGGATTG
1336

2579
T

TTAAATTCA

HVP58
58_sp_1320_
AAAATTATTGAGCTAGAAGACAGCGGA
1337
TGATATTTCCTCCATCGTTTTC
1338

2706
T

CTTGTC

HVP58
58_fus_2867_
GCAGGACAAAATCCTAGACATATACGA
1339
AAATACGGCTGCACCGAGT
1340

MYC_001_
A

exon2

HVP58
58_sp_232_532
GTCAGGCGTTGGAGACATCT
1341
GCGTTGGGTTGTTTCCTCTCA
1342

HVP58
58_fus_898_
TGCTTATGGGCACATGTACCATT
1343
CATGGTTCCACCAGCGTTATT
1344

PVT1_004_

exon1

HVP58
58_fus_898_
TGCTTATGGGCACATGTACCATT
1345
CTGAGAAGCCCTGCCCTTC
1346

MYC_001_

exon1

HVP58
58_sp_898_2706
TGCTTATGGGCACATGTACCATT
1347
TGATATTTCCTCCATCGTTTTC
1348

CTTGTC

HVP58
58_fus_3596_
GAGGAGGACTACACAGTACAACTAACT
1349
TCTTTGCTCGCAGCTCGT
1350

PVT1_005_

exon1

HVP58
58_fus_2867_
GCAGGACAAAATCCTAGACATATACGA
1351
CATGGTTCCACCAGCGTTATT
1352

PVT1_004_
A

exon1

HVP58
58_sp_232_3355
GTCAGGCGTTGGAGACATCT
1353
CCCTGTGTACTTTCGTTGTTGGT
1354

HVP58
58_fus_2867_
GCAGGACAAAATCCTAGACATATACGA
1355
ATCATGATGGCTGTATGTGCCA
1356

PVT1_002_
A

exon3

HVP58
58_fus_3596_
GAGGAGGACTACACAGTACAACTAACT
1357
AAATACGGCTGCACCGAGT
1358

MYC_001_

exon2

HVP58
58_unsp_3608_
GAGGAGGACTACACAGTACAACTAACT
1359
CCAATGCCATGTGGATGACATAT
1360

3609

TACA

HVP58
58_unsp_5642_
CTGATTTTATGTTGCACCCTAGCTATTT
1361
GCTTACAACCTTAGACACAGGC
1362

5643

A

HVP58
58_fus_3596_
GAGGAGGACTACACAGTACAACTAACT
1363
CTGAGAAGCCCTGCCCTTC
1364

MYC_001_

exon1

HVP58
58_unsp_232_
GTCAGGCGTTGGAGACATCT
1365
TCGTAAGCACACTTTACATACTG
1366

233

CAAA

HVP58
58_sp_898_2579
TGCTTATGGGCACATGTACCATT
1367
TGCATCAAATGGAAATGGATTG
1368

TTAAATTCA

HVP58
58_sp_898_3355
TGCTTATGGGCACATGTACCATT
1369
CCCTGTGTACTTTCGTTGTTGGT
1370

HVP58
58_sp_3608_
GAGGAGGACTACACAGTACAACTAACT
1371
GCTTACAACCTTAGACACAGGC
1372

5643

A

HVP58
58_fus_2867_
GCAGGACAAAATCCTAGACATATACGA
1373
CTGAGAAGCCCTGCCCTTC
1374

MYC_001_exon1
A

HVP58
58_gen_5893_7295
CGTTTGGTCTGGGCATGTGTA
1375
GCTGTGCGGGATATCTGTTACTG
1376

HVP58
58_gen_38S8_4208
TCTATATATGCTTGGTTGCTGGTGTTG
1377
CATGTGCAGAACCAGTATACAG
1378

TTAGT

HVP58
58_gen_1570_2329
CAATGGGACAATGGATACAAAGTAGGT
1379
GGGCCACACAGTAACATACAAC
1380

T

HVP58
58_gen_7302_7824
TCTATGAGTAAGGTGCTGTCCCTAAAT
1381
GGAGGTAAAGTAAAATGGAGG
1382

CAGTA

HVP59
59_unsp_3627_3628
TCCGTTTGCATCCAGGCAA
1383
CCAATGCCAGGTAGAGGAAATA
1384

TTTTCA

HVP59
59_fus_2862_
GCGTTTAAGTGTGTTACAGGATCAAAT
1385
CTGAGAAGCCCTGCCCTTC
1386

MYC_001_exon1

HVP59
59_sp_1306_3359
AAAGAAGGTTAATAACAGTGCCAGACA
1387
CCCAAGTACGTGGCTTCGG
1388

HVP59
59_unsp_2570_2571
AGATAGAAAGCATAGGCACCTAGTACA
1389
TCTATTTTTGTCAAATGGCAAT
1390

A

TTGTTTGGA

HVP59
59_unsp_748_749
CAGATGGAGTTAATCATCCTTTGCTACT
1391
TGTAAGGCTCGCAATCCGT
1392

HVP59
59_fus_887_
ACTATCCTTTGTGTGTCCTTTGTGT
1393
CATGGTTCCACCAGCGTTATT
1394

PVT1_004_exon1

HVP59
59_fus_2862_
GCGTTTAAGTGTGTTACAGGATCAAAT
1395
CATGGTTCCACCAGCGTTATT
1396

PVT1_004_exon1

HVP59
59_fus_887_
ACTATCCTTTGTGTGTCCTTTGTGT
1397
ATCATGATGGCTGTATGTGCCA
1398

PVT1_002_exon3

HVP59
59_fus_3615_
TCCGTTTGCATCCAGGCAA
1399
CTGAGAAGCCCTGCCCTTC
1400

MYC_001_exon1

HVP59
59_sp_1306_2698
AAAGAAGGTTAATAACAGTGCCAGACA
1401
GGTGTCCATCACTGTCTGCAT
1402

HVP59
59_sp_3627_5606
TCCGTTTGCATCCAGGCAA
1403
TGACATACTCATCAGTGCTGA
1404

CAAC

HVP59
59_fus_3615_
TCCGTTTGCATCCAGGCAA
1405
AAATACGGCTGCACCGAGT
1406

MYC_001_exon2

HVP59
59_fus_2862_
GCGTTTAAGTGTGTTACAGGATCAAAT
1407
GGTGATCCAGACTCTGACCT
1408

MYC_001_exon3

TTTG

HVP59
59_unsp_5768_5769
GTATGTCACCCGTACCAGTATTTTCTAC
1409
GCCAAATTTATTGGGATCAGGT
1410

AACTT

HVP59
59_sp_887_3359
ACTATCCTTTGTGTGTCCTTTGTGT
1411
CCCAAGTACGTGGCTTCGG
1412

HVP59
59_sp_183_2571
GCATCAAHGTGTGTTTTGCAAAGG
1413
TCTATTTTTGTCAAATGGCAAT
1414

TTGTTTGGA

HVP59
59_fus_3615_
TCCGTTTGCATCCAGGCAA
1415
GGTGATCCAGACTCTGACCTTTT
1416

MYC_001_exon3

G

HVP59
59_fus_887_
ACTATCCTTTGTGTGTCCTTTGTGT
1417
GGTGATCCAGACTCTGACCTTTT
1418

MYC_001_exon3

G

HVP59
59_fus_3615_
TCCGTTTGCATCCAGGCAA
1419
TCTTTGCTCGCAGCTCGT
1420

PVT1_005_exon1

HVP59
59_fus_2862_
GCGTTTAAGTGTGTTACAGGATCAAAT
1421
AAATACGGCTGCACCGAGT
1422

MYC_001_exon2

HVP59
59_sp_887_5606
ACTATCCTTTGTGTGTCCTTTGTGT
1423
TGACATACTCATCAGTGCTGACA
1424

AC

HVP59
59_fus_887_
ACTATCCTTTGTGTGTCCTTTGTGT
1425
AAATACGGCTGCACCGAGT
1426

MYC_001_exon2

HVP59
59_sp_887_5769
ACTATCCTTTGTGTGTCCTTTGTGT
1427
GCCAAATTTATTGGGATCAGGT
1428

AACTT

HVP59
59_unsp_887_888
ACTATCCTTTGTGTGTCCTTTGTGT
1429
CGTCATCTGAAATTTTGTCACCT
1430

GTTTT

HVP59
59_fus_2862_
GCGrTTAAGTGTGTTACAGGATCAAAT
1431
ATCATGATGGCTGTATGTGCCA
1432

PVT1_002_exon3

HVP59
S9_sp_3627_
TCCGTTTGCATCCAGGCAA
1433
GCCAAATTTATTGGGATCAGGT
1434

5769

AACTT

HVP59
S9_sp_1306_
AAAGAAGGTTAATAACAGTGCCAGACA
1435
TCTATTTTTGTCAAATGGCAAT
1436

2571

TTGTTTGGA

HVP59
59_sp_887_2571
ACTATCCTTTGTGTGTCCTTTGTGT
1437
TCTATTTTTGTCAAATGGCAAT
1438

TTGTTTGGA

HVP59
59_unsp_1306_
AAAGAAGGTTAATAACAGTGCCAGACA
1439
GTCTATTTGACTGTCGCTACAAA
1440

1307

CAC

HVPS9
59_unsp_5605_
CCTCGTAAACGTAAACGTGTTCC
1441
TGACATACTCATCAGTGCTGACA
1442

5606

AC

HVP59
59_fus_3615_
TCCGTTTGCATCCAGGCAA
1443
ATCATGATGGCTGTATGTGCCA
1444

PVT1_002_

exon3

HVP59
59_fus_3615_
TCCGTTTGCATCCAGGCAA
1445
CATGGTTCCACCAGCGTTATT
1446

PVT1_004_

exon1

HVP59
59_fus_2862_
GCGTTTAAGTGTGTTACAGG ATCAAAT
1447
TCTTTGCTCGCAGCTCGT
1448

PVT1_005_

exon1

HVP59
59_sp_183_3359
GCATCAATTGTGTGTTTTGCAAAGG
1449
CCCAAGTACGTGGCTTCGG
1450

HVP59
59_sp_887_2698
ACTATCCTTTGTGTGTCCTTTGTGT
1451
GGTGTCCATCACTGTCTGCAT
1452

HVP59
59_sp_183_2698
GCATCAATTGTGTGTTTTGCAAAGG
1453
GGTGTCCATCACTGTCTGCAT
1454

HVP59
59_fus_887_
ACTATCCTTTGTGTGTCCTTTGTGT
1455
CTGAGAAGCCCTGCCCTTC
1456

MYC_001_exon1

HVP59
59_fus_887_
ACTATCCTTTGTGTGTCCTTTGTGT
1457
TCTTTGCTCGCAGCTCGT
1458

PVT1_005_

exon1

HVP59
59_sp_183_749
GCATCAATTGTGTGTTTTGCAAAGG
1459
TGTAAGGCTCGCAATCCGT
1460

HVP59
59_unsp_183_184
GCATCAATTGTGTGTTTTGCAAAGG
1461
GCATTTCAGACACGCTGCATAC
1462

HVP59
59_gen_3877_4222
GTTGCAATGTCCCGCTTCTG
1463
CATGGGCATATAGTAGTAACAG
1464

TGGAA

HVP59
59_gen_7261_7896
GGTTGCACCCAATGAGTAAGGTA
1465
GCAAAACTGGACATTCAGGACA
1466

AAA

HVP59
59_gen_6019_7254
GCTGTGTACCTGCCATTGGA
1467
CTGTGTCTACCATATCACCATC
1468

TTCA

HVP59
59_gen_1556_2321
GTGCATGTTAATTGAACCACCCAAA
1469
TCAAACACGCTATCATCAACTCC
1470

AT

HVP66
66_fus_2843_
CGTGCCAGAACAAAATACTAGACTGT
1471
ATCATGATGGCTGTATGTGCCA
1472

PVT1_002_

exon3

HVP66
66_sp_233_773
CACCATCTGAGCGAGGTATTACA
1473
TGTACCACACGTAGCTCCTCT
1474

HVP66
66_fus_910_
GTTAACAGTAACGTGCCCACTCT
1475
CTGAGAAGCCCTGCCCTTC
1476

MYC_001_exon1

HVP66
66_unsp_233_
CACCATCTGAGCGAGGTATTACA
1477
ACAATAAACATACCCTACATACT
1478

234

GCATATGG

HVP66
66_fus_910_
GTTAACAGTAACGTGCCCACTCT
1479
GGTGATCCAGACTCTGACCTTTT
1480

MYC_001_exon3

G

HVP66
66_fus_2843_
CGTGCCAGAACAAAATACTAGACTGT
1481
CTGAGAAGCCCTGCCCTTC
1482

MYC_001_

exon1

HVP66
66_sp_1290_
GAAGACAGCGGGTATGGCAATA
1483
TTTTCTTTGTCCTCGTCGTT
1484

2682

ATCCAA

HVP66
66_fus_2843_
CGTGCCAGAACAAAATACTAGACTGT
1485
GGTGATCCAGACTCTGACCTTTT
1486

MYC_001_

G

exon3

HVP66
66_sp_1290_
GAAGACAGCGGGTATGGCAATA
1487
CATTACTTAATTCATACACAGGA
1488

2555

TTACCATT

HVP66
66_fus_3605_
GTATCAACACACAAAGCCACTGT
1489
TCTTTGCTCGCAGCTCGT
1490

PVT1_005_

exon1

HVP66
66_sp_910_5647
GTTAACAGTAACGTGCCCACTCT
1491
ACAACCTTTGAAACAGGTGTTG
1492

GA

HVP66
66_fus_3605_
GTATCAACACACAAAGCCACTGT
1493
GGTGATCCAGACTCTGACCTTTT
1494

MYC_001_exon3

G

HVP66
66_sp_910_3362
GTTAACAGTAACGTGCCCACTCT
1495
GGTGGTGGTGGTCCTGTG
1496

HVP66
66_fus_3605_
GTATCAACACACAAAGCCACTGT
1497
CATGGTTCCACCAGCGTTATT
1498

PVT1_004_exon1

HVP66
66_fus_2843_
CGTGCCAGAACAAAATACTAGACTGT
1499
AAATACGGCTGCACCGAGT
1500

MYC_001_exon2

HVP66
66_sp_233_2682
CACCATCTGAGCGAGGTATTACA
1501
TTTTCTTTGTCCTCGTCGTTA
1502

TCCAA

HVP66
66_sp_233_533
CACCATCTGAGCGAGGTATTACA
1503
AACCTCTTGCAACGTTGGTACT
1504

HVP66
66_unsp_772_773
ACAGCAAGCTAGACAAGCTGAA
1505
TGTACCACACGTAGCTCCTCT
1506

HVP66
66_sp_233_416
CACCATCTGAGCGAGGTATTACA
1507
GAAATCGTCTTTTATGTTCACAG
1508

TGCAA

HVP66
66_sp_233_2555
CACCATCTGAGCGAGGTATTACA
1509
CATTACTTAATTCATACACAGGA
1510

TTACCATT

HVP66
66_sp_3617_5647
GTATCAACACACAAAGCCACTGT
1511
ACAACCTTTGAAACAGGTGTTG
1512

GA

HVP66
66_fus_2843_
CGTGCCAGAACAAAATACTAGACTGT
1513
CATGGTTCCACCAGCGTTATT
1514

PVT1_004_exon1

HVP66
66_fus_910_
GTTAACAGTAACGTGCCCACTCT
1515
ATCATGATGGCTGTATGTGCCA
1516

PVT1_002_

exon3

HVP66
66_unsp_910_911
GTTAACAGTAACGTGCCCACTCT
1517
TTCTACAATTGCTTCTACCTGAA
1518

ACCAT

HVP66
66_sp_233_3362
CACCATCTGAGCGAGGTATTACA
1519
GGTGGTGGTGGTCCTGTG
1520

HVP66
66_sp_910_2555
GTTAACAGTAACGTGCCCACTCT
1521
CATTACTTAATTCATACACAGGA
1522

TTACCATT

HVP66
66_fus_3605_
GTATCAACACACAAAGCCACTGT
1523
AAATACGGCTGCACCGAGT
1524

MYC_001_exon2

HVP66
66_fus_2843_
CGTGCCAGAACAAAATACTAGACTGT
1525
TCTTTGCTCGCAGCTCGT
1526

PVT1_005_exon1

HVP66
66_fus_3605_
GTATCAACACACAAAGCCACTGT
1527
ATCATGATGGCTGTATGTGCCA
1528

PVT1_002_exon3

HVP66
66_sp_1290_3362
GAAGACAGCGGGTATGGCAATA
1529
GGTGGTGGTGGTCCTGTG
1530

HVP66
66_sp_910_2682
GTTAACAGTAACGTGCCCACTCT
1531
TTTTCTTTGTCCTCGTCGTTA
1532

TCCAA

HVP66
66_fus_910_
GTTAACAGTAACGTGCCCACTCT
1533
AAATACGGCTGCACCGAGT
1534

MYC_001_exon2

HVP66
66_fus_3605_
GTATCAACACACAAAGCCACTGT
1535
CTGAGAAGCCCTGCCCTTC
1536

MYC_001_exon1

HVP66
66_unsp_1290_1291
GAAGACAGCGGGTATGGCAATA
1537
GATACCGAGTGCTCACTACAATT
1538

ACTG

HVP66
66_fus_910_
GTTAACAGTAACGTGCCCACTCT
1539
CATGGTTCCACCAGCGTTATT
1540

PVT1_004_exon1

HVP66
66_fus_910_
GTTAACAGTAACGTGCCCACTCT
1541
TCTTTGCTCGCAGCTCGT
1542

PVT1_005_exon1

HVP66
66_unsp_3617_3618
GTATCAACACACAAAGCCACTGT
1543
TCTGTACTTGTCCAATGATATGT
1544

TGTTGT

HVP66
66_unsp_5646_5647
GCTACATTTGCACTATGGCCTGTA
1545
ACAACCTTTGAAACAGGTGTTG
1546

GA

HVP66
66_unsp_415_416
GGGCAACATTAGAAAGTATAACTAAAA
1547
GAAATCGTCTTTTATGTTCACAG
1548

AACA

TGCAA

HVP66
66_gen_7301_7824
GGTTAGGTGGTGTTCCTTACTGTTTA
1549
CAAAAGGCTAGGCAACCGAATT
1550

HVP66
66_gen_1540_2305
AGACATAGATAGCAATGCACAAGCA
1551
ATCACCCCCTTCATCTACTTT
1552

ACTACA

HVP66
66_gen_3867_4235
GTTTGTCTGTGTGTGTGCCATT
1553
GCATGGCAATATATACACAGTG
1554

TAGGT

HVP66
66_gen_5897_7294
GTAGGCCGAGGTCAACCTTTA
1555
GTGCACATCCCACAATACATAAC
1556

TG

HVP66
66_sp_1290_3331
GACAGGGAGACAGCTCAACAATTATT
1557
CTCTCGGTACACAGTTTGCTGAT
1558

TA

HVP66
66_unsp_3330_3331
GTGGGTGGTGTAAAGTGTCATCA
1559
GGACAGTAAATACTCTCGGTTTC
1560

CAT

HVP68
68_sp_129_3292
GACATTGGACACTACATTGCATGAC
1561
TCGCGGTGGTGTTCTGTAG
1562

HVP68
68_sp_129_2510
GACATTGGACACTACATTGCATGAC
1563
CTGTTTTGGTCAAATGGAAATGC
1564

ATTAG

HVP68
68_fus_2801_
ACAGGACAGTAAATGTATACAGGACCA
1565
CTGAGAAGCCCTGCCCTTC
1566

MYC_001_exon1
T

HVP68
68_unsp_5487_5488
TACAACCTTTGCCATAACTATATATGGT
1567
ATTGACAACCTTCGCCACTGA
1568

HVP68
68_fus_3551_
AGTAGAAGTGCAGGCCAAAACAA
1569
CATGGTTCCACCAGCGTTATT
1570

PVT1_004_exon1

HVP68
68_fus_838_
TCCGTGGTGTGCAACTGAA
1571
CTGAGAAGCCCTGCCCTTC
1572

MYC_001_exon1

HVP68
68_fus_3551_
AGTAGAAGTGCAGGCCAAAACAA
1573
GGTGATCCAGACTCTGACCTTTT
1574

MYC_001_exon3

G

HVP68
68_fus_2801_
ACAGGACAGTAAATGTATACAGGACCA
1575
ATCATGATGGCTGTATGTGCCA
1576

PVT1_002_exon3
T

HVP68
68_fus_2801_
ACAGGACAGTAAATGTATACAGGACCA
1577
AAATACGGCTGCACCGAGT
1578

MYC_001_exon2
T

HVP68
68_fus_838_
TCCGTGGTGTGCAACTGAA
1579
CATGGTTCCACCAGCGTTATT
1580

PVT1_004_exon1

HVP68
68_unsp_1233_1234
AGACAACCGGCGTATACAGTG
1581
CACACTACTACAGTCCTCCCGTA
1582

T

HVP68
68_fus_3551_
AGTAGAAGTGCAGGCCAAAACAA
1583
CTGAGAAGCCCTGCCCTTC
1584

MYC_001_exon1

HVP68
68_fus_2801_
ACAGGACAGTAAATGTATACAGGACCA
1585
CATGGTTCCACCAGCGTTATT
1586

PVT1_004_exon1
T

HVP68
68_unsp_129_130
GACATTGGACACTACATTGCATGAC
1587
GATTGGCATGCAGCAAATGGTA
1588

HVP68
68_fus_2801_
ACAGGACAGTAAATGTATACAGGACCA
1589
GGTGATCCAGACTCTGACCTTTT
1590

MYC_001_exon3
T

G

HVP68
68_sp_1233_2510
AGACAACCGGCGTATACAGTG
1591
CTGTTTTGGTCAAATGGAAATGC
1592

ATTAG

HVP68
68_unsp_696_697
CCACCAACATCTACTACTAGCCAGA
1593
CTGTTGTAGTGTCCGCAGGTT
1594

HVP68
68_unsp_2509_2510
CCTAATACAAATAAAGTGTCCACCAAT
1595
CTGTTTTGGTCAAATGGAAATGC
1596

GCT

ATTAG

HVP68
68_sp_129_312
GACATTGGACACTACATTGCATGAC
1597
CTTCGTTTTGTTGTTAGGTGCCT
1598

TAG

HVP68
68_fus_3551_
AGTAGAAGTGCAGGCCAAAACAA
1599
ATCATGATGGCTGTATGTGCCA
1600

PVT1_002_exon3

HVP68
68_fus_3551_
AGTAGAAGTGCAGGCCAAAACAA
1601
TCTTTGCTCGCAGCTCGT
1602

PVT1_005_exon1

HVP68
68_sp_838_2510
TCCGTGGTGTGCAACTGAA
1603
CTGTTTTGGTCAAATGGAAATGC
1604

ATTAG

HVP68
68_unsp_838_839
TCCGTGGTGTGCAACTGAA
1605
GACTGTGTCACCTGTTTGTTTAT
1606

CTACT

HVP68
68_sp_838_5488
TCCGTGGTGTGCAACTGAA
1607
ATTGACAACCTTCGCCACTGA
1608

HVP68
68_fus_3551_
AGTAGAAGTGCAGGCCAAAACAA
1609
AAATACGGCTGCACCGAGT
1610

MYC_001_exon2

HVP68
68_fus_838_
TCCGTGGTGTGCAACTGAA
1611
ATCATGATGGCTGTATGTGCCA
1612

PVT1_002_exon3

HVP68
68_fus_838_
TCCGTGGTGTGCAACTGAA
1613
TCTTTGCTCGCAGCTCGT
1614

PVT1_005_exon1

HVP68
68_sp_838_3292
TCCGTGGTGTGCAACTGAA
1615
TCGCGGTGGTGTTCTGTAG
1616

HVP68
68_sp_3563_5488
AGTAGAAGTGCAGGCCAAAACAA
1617
ATTGACAACCTTCGCCACTGA
1618

HVP68
68_fus_838_
TCCGTGGTGTGCAACTGAA
1619
AAATACGGCTGCACCGAGT
1620

MYC_001_exon2

HVP68
68_sp_129_697
GACATTGGACACTACATTGCATGAC
1621
CTGTTGTAGTGTCCGCAGGTT
1622

HVP68
68_unsp_3291_3292
CTAGTGGAAAATGGGACGTGCATTATA
1623
TCGCGGTGGTGTTCTGTAG
1624

HVP68
68_unsp_3563_3564
AGTAGAAGTGCAGGCCAAAACAA
1625
AAGCGTTATGTTTTTGCAACCTA
1626

TACC

HVP68
68_sp_1233_3292
AGACAACCGGCGTATACAGTG
1627
TCGCGGTGGTGTTCTGTAG
1628

HVP68
68_fus_838_
TCCGTGGTGTGCAACTGAA
1629
GGTGATCCAGACTCTGACCTTTT
1630

MYC_001_exon3

G

HVP68
68_fus_2801_
ACAGGACAGTAAATGTATACAGGACCA
1631
TCTTTGCTCGCAGCTCGT
1632

PVT1_005_exon1
T

HVP68
68_unsp_311_312
GGAATCGGTGTATGCAACTACATTA6A
1633
CTTCGTTTTGTTGTTAGGTGCCT
1634

A

TAG

HVP68
68_gen_7154_7822
CCCTGTGACTAACATATGTCCTTGT
1635
CCACACGGTATAGTTTGCAACCA
1636

T

HVP68
68_gen_5738_7147
GCCTGTGTTGGTGTTGAAATAGGTA
1637
TGCAACATTGTCCCTACTGTCTTT
1638

AG

HVP68
68_gen_3813_4090
GGTGTGGTTTTGTGTATGCATGT
1639
GGTATACAGCAAACACCTCAAA
1640

TGGT

HVP68
68_gen_1483_2260
CGACACGCCGGAATG6ATAA
1641
CGCTGCAGCATTACTATTACAAT
1642

CTG

HVP73
73_unsp_5493_5494
TGGGTCAGGTTTTATATTACACCCTAGT
1643
GCTTACAACCTTAGACACAGACA
1644

CA

HVP73
73_fus_2858_
GTATGAACGTGACAGTGTACACCTAA
1645
CTGAGAAGCCCTGCCCTTC
1646

MYC_001_exon1

HVP73
73_sp_1287_3346
AAACGAAGACTGTTTGAGGAGCA
1647
TGGTGTTGGTGGTTGTGGT
1648

HVP73
73_fus_3560_
ACCTACATCCCACCACAGAGT
1649
AAATACGGCTGCACCGAGT
1650

MYC_001_exon2

HVP73
73_sp_862_2570
TGCTTATGGGTACACTAGGTATTGTGT
1651
GGGTTCCCATTACTGTCAAATGG
1652

A

HVP73
73_fus_862_MYC_
TGCTTATGGGTACACTAGGTATTGTGT
1653
GGTGATCCAGACTCTGACCTTTT
1654

001_exon3

G

HVP73
73_sp_227_410
AGCGTTATGTGACGAAGTGAATATTTC
1655
CTGTTCTGCTATTTGATGAAACC
1656

T

GTTTT

HVP73
73_sp_227_527
AGCGTTATGTGACGAAGTGAATATTTC
1657
TTCGGTTGTTGGTTTCAGGTCTA
1658

T

A

HVP73
73_sp_862_3346
TGCTTATGGGTACACTAGGTATTGTGT
1659
TGGTGTTGGTGGTTGTGGT
1660

HVP73
73_unsp_3345_3346
GGGTAAAAGGCATATGGGAAGTACAT
1661
TGGTGTTGGTGGTTGTGGT
1662

HVP73
73_unsp_2569_2570
CAAGTTAAATGCCCTCCATTACTGATAA
1663
GGGTTCCCATTACTGTCAAATGG
1664

C

A

HVP73
73_fus_862_
TGCTTATGGGTACACTAGGTATTGTGT
1665
CATGGTTCCACCAGCGTTATT
1666

PVT1_004_exon1

HVP73
73_unsp_3572_
ACCTACATCCCACCACAGAGT
1667
GTCCAATGCCATGTTGTTGTTAC
1668

3573

A

HVP73
73_fus_3560_
ACCTACATCCCACCACAGAGT
1669
ATCATGATGGCTGTATGTGCCA
1670

PVT1_002_exon3

HVP73
73_sp_862_5647
TGCTTATGGGTACACTAGGTATTGTGT
1671
ACGAAGCCTAAACACCCTGTATT
1672

G

HVP73
73_fus_2858_
GTATGAACGTGACAGTGTACACCTAA
1673
ATCATGATGGCTGTATGTGCCA
1674

PVT1_002_exon3

HVP73
73_unsp_726_727
ACTCAGAGGATGAGGATGAAACAGA
1675
CCTAGTGTACCCATAAGCAACTC
1676

TTCTA

HVP73
73_sp_3572_5494
ACCTACATCCCACCACAGAGT
1677
GCTTACAACCTTAGACACAGACA
1678

CA

HVP73
73_unsp_1287_1288
AAACGAAGACTGTTTGAGGAGCA
1679
GACACAATTTGGTTGCCTTCTTC
1680

ATTAA

HVP73
73_fus_862_MYC_
TGCTTATGGGTACACTAGGTATTGTGT
1681
AAATACGGCTGCACCGAGT
1682

001_exon2

HVP73
73_unsp_5646_5647
TGCAGGTAGCACACGTTTGT
1683
ACGAAGCCTAAACACCCTGTATT
1684

G

HVP73
73_fus_3560_PVT1_
ACCTACATCCCACCACAGAGT
1685
TCTTTGCTCGCAGCTCGT
1686

005_exon1

HVP73
73_unsp_862_863
TGCTTATGGGTACACTAGGTATTGTGT
1687
TGGAATTGGATCCCCTGTTTTTC
1688

TTT

HVP73
73_fus_862_
TGCTTATGGGTACACTAGGTATTGTGT
1689
TCTTTGCTCGCAGCTCGT
1690

PVT1_005_exon1

HVP73
73_fus_862_MYC_
TGCTTATGGGTACACTAGGTATTGTGT
1691
CTGAGAAGCCCTGCCCTTC
1692

001_exon1

HVP73
73_sp_862_5494
TGCTTATGGGTACACTAGGTATTGTGT
1693
GCTTACAACCTTAGACACAGACA
1694

CA

HVP73
73_fus_2858_
GTATGAACGTGACAGTGTACACCTAA
1695
CATGGTTCCACCAGCGTTATT
1696

PVT1_004_exon1

HVP73
73_unsp_227_228
AGCGTTATGTGACGAAGTGAATATTTC
1697
AAAATTTTAAACACGGTTGACAT
1698

T

ACAC

HVP73
73_fus_28S8_MYC_
GTATGAACGTGACAGTGTACACCTAA
1699
AAATACGGCTGCACCGAGT
1700

001_exon2

HVP73
73_fus_2858_
GTATGAACGTGACAGTGTACACCTAA
1701
TCTTTGCTCGCAGCTCGT
1702

PVT1_005_exon1

HVP73
73_fus_862_PVT1_
TGCTTATGGGTACACTAGGTATTGTGT
1703
ATCATGATGGCTGTATGTGCCA
1704

002_exon3

HVP73
73_unsp_409_410
AGACAATCAGTATATGGCACTACGTTA
1705
CTGTTCTGCTATTTGATGAAACC
1706

GA

GTTTT

HVP73
73_fus_3560_PVT1_
ACCTACATCCCACCACAGAGT
1707
CATGGTTCCACCAGCGTTATT
1708

004_exon1

HVP73
73_sp_227_2570
AGCGTTATGTGACGAAGTGAATATTTC
1709
GGGTTCCCATTACTGTCAAATGG
1710

T

A

HVP73
73_fus_3560_MYC_
ACCTACATCCCACCACAGAGT
1711
CTGAGAAGCCCTGCCCTTC
1712

001_exon1

HVP73
73_fus_3560_MYC_
ACCTACATCCCACCACAGAGT
1713
GGTGATCCAGACTCTGACCTTTT
1714

001_exon3

G

HVP73
73_sp_1287_2570
AAACGAAGACTGTTTGAGGAGCA
1715
GGGTTCCCATTACTGTCAAATGG
1716

A

HVP73
73_sp_227_727
AGCGTTATGTGACGAAGTGAATATTTC
1717
CCTAGTGTACCCATAAGCAACTC
1718

T

TTCTA

HVP73
73_sp_227_3346
AGCGTTATGTGACGAAGTGAATATTTC
1719
TGGTGTTGGTGGTTGTGGT
1720

T

HVP73
73_fus_2858_MYC_
GTATGAACGTGACAGTGTACACCTAA
1721
GGTGATCCAGACTCTGACCTTTT
1722

001_exon3

G

HVP73
73_sp_3572_5647
ACCTACATCCCACCACAGAGT
1723
ACGAAGCCTAAACACCCTGTATT
1724

G

HVP73
73_gen_3822_4053
TCGCTTGCAGTGTCTGTGTATATTT
1725
CATGGTAATGTACAAGTGCCAT
1726

AGGA

HVP73
73_gen_1537_2320
GAACGCATGTTAATTGAACCTCCAA
1727
GCTGCACTAACGTTTGTCTTTTA
1728

ATCC

HVP73
73_gen_5897_7198
TGTATTTTAGGTTGTAGGCCTCCCTTA
1729
CTCCAAAGCCAACATCTATCATA
1730

TCAC

HVP73
73_gen_7205_7700
GTCGCCATTTTACATGCATTAAGGT
1731
AGGAAACAAACCCTGCCAAGTT
1732

HVP82
82_sp_3613_5571
TGCGACCACCAAATACACTGT
1733
GTGTTGACAATGCGTGACACT
1734

HVP82
82_fus_3601_MYC_
TGCGACCACCAAATACACTGT
1735
CTGAGAAGCCCTGCCCTTC
1736

001_exon1

HVP82
82_fus_2860_MYC_
GTGCCAGGAGAAAATACTAGA
1737
GGTGATCCAGACTCTGACCTTTT
1738

001_exon3
CTGTTAT

G

HVP82
82_fus_2860_PVT1_
GTGCCAGGAGAAAATACTAGAC
1739
TCTTTGCTCGCAGCTCGT
1740

005_exon1
TGTTAT

HVP82
82_fus_2860_MYC_
GTGCCAGGAGAAAATACTAGAC
1741
CTGAGAAGCCCTGCCCTTC
1742

001_exon1
TGTTAT

HVP82
82_unsp_1316_1317
CCGGACAGTGGATATGGCAATA
1743
GGTCTATCTCTGTACTTCTGTCG
1744

CT

HVP82
82_sp_222_753
CCTGCAATACGTCTATGCACAAT
1745
CCAGTAACATTTGCTGAAATATG
1746

CGAA

HVP82
82_unsp_752_753
GGAGGATGAAGTAGATAATATGCGTG
1747
CCAGTAACATTTGCTGAAATATG
1748

AC

CGAA

HVP82
82_unsp_3344_3345
GGGCACAACAATGGGAGGTA
1749
GGGTGTTCGATAGCTGTTCAA
1750

HVP82
82_unsp_3613_3614
TGCGACCACCAAATACACTGT
1751
CAATGCCAGGTAGATGACACTT
1752

CTTTAA

HVP82
82_unsp_5570_5571
GGGATTACTACTTTGTGGCCGTATA
1753
GTGTTGACAATGCGTGACACT
1754

HVP82
82_sp_222_407
CCTGCAATACGTCTATGCACAAT
1755
TTTTTTGTCGTCCACCACCT
1756

TTTG

HVP82
82_fus_3601_
TGCGACCACCAAATACACTGT
1757
GGTGATCCAGACTCTGACCT
1758

MYC_001_exon3

TTTG

HVP82
82_fus_888_MYC_
CGTGGTGTGCGACCAACTAA
1759
AAATACGGCTGCACCGAGT
1760

001_exon2

HVP82
82_fus_888_MYC_
CGTGGTGTGCGACCAACTAA
1761
CTGAGAAGCCCTGCCCTTC
1762

001_exon1

HVP82
82_sp_222_3345
CCTGCAATACGTCTATGCACAAT
1763
GGGTGTTCGATAGCTGTTCAA
1764

HVP82
82_unsp_222_223
CCTGCAATACGTCTATGCACAAT
1765
CATGCTGCATATGGCGTATTGTC
1766

HVP82
82_unsp_406_407
GTAGGTCTGTGTATGGTGCTACATT
1767
TTTTTTGTCGTCCACCACC
1768

TTTTG

HVP82
82_fus_3601_
TGCGACCACCAAATACACTGT
1769
ATCATGATGGCTGTATGTGCCA
1770

PVT1_002_exon3

HVP82
82_fus_3601_
TGCGACCACCAAATACACTGT
1771
TCTTTGCTCGCAGCTCGT
1772

PVT1_005_exon1

HVP82
82_sp_1316_
CCGGACAGTGGATATGGCAATA
1773
GGGTGTTCGATAGCTGTTCAA
1774

3345

HVP82
82_sp_888_5571
CGTGGTGTGCGACCAACTAA
1775
GTGTTGACAATGCGTGACACT
1776

HVP82
82_fus_888_
CGTGGTGTGCGACCAACTAA
1777
CATGGTTCCACCAGCGTTATT
1778

PVT1_004_exon1

HVP82
82_sp_888_2575
CGTGGTGTGCGACCAACTAA
1779
CATCATTTAGTGCATATACAGGA
1780

TTCCC

HVP82
82_sp_888_3345
CGTGGTGTGCGACCAACTAA
1781
GGGTGTTCGATAGCTGTTCAA
1782

HVP82
82_fus_2860_
GTGCCAGGAGAAAATACTAGA
1783
CATGGTTCCACCAGCGTTATT
1784

PVT1_004_exon1
CTGTTAT

HVP82
82_sp_1316_
CCGGACAGTGGATATGGCAATA
1785
CATCATTTAGTGCATATACAGGA
1786

2575

TTCCC

HVP82
82_fus_3601_
TGCGACCACCAAATACACTGT
1787
CATGGTTCCACCAGCGTTATT
1788

PVT1_004_exon1

HVP82
82_fus_888_
CGTGGTGTGCGACCAACTAA
1789
TCTTTGCTCGCAGCTCGT
1790

PVT1_005_exon1

HVP82
82_sp_222_
CCTGCAATACGTCTATGCACAAT
1791
CATCATTTAGTGCATATACAGGA
1792

2575

TTCCC

HVP82
82_fus_888_
CGTGGTGTGCGACCAACTAA
1793
GGTGATCCAGACTCTGACC
1794

MYC_001_exon3

TTTTG

HVP82
82_unsp_
CGTGGTGTGCGACCAACTAA
1795
TTGTCAACTACTGCCTCCA
1796

888_889

CATAAAA

HVP82
82_sp_
CCTGCAATACGTCTATGCACAAT
1797
TCCAACACTATGTCCTTTA
1798

222_521

ATTGTGGT

HVP82
82_fus_3601_
TGCGACCACCAAATACACTGT
1799
AAATACGGCTGCACCGAGT
1800

MYC_001_exon2

HVP82
82_fus_2860_
GTGCCAGGAGAAAATACTAG
1801
AAATACGGCTGCACCGAGT
1802

MYC_001_exon2
ACTGTTAT

HVP82
82_fus_2860_
GTGCCAGGAGAAAATACTAGA
1803
ATCATGATGGCTGTATGTGCCA
1804

PVT1_002_
CTGTTAT

exon3

HVP82
82_fus_888_
CGTGGTGTGCGACCAACTAA
1805
ATCATGATGGCTGTATGTGCCA
1806

PVT1_002_

exon3

HVP82
82_unsp_
ACACAGAAGCCTGCTGCAAA
1807
CATCATTTAGTGCATATACAGGA
1808

2574_2575

TTCCC

HVP82
82_gen_
CCTGTAGGTTAAGGGTGGTGTT
1809
AAATCGGTCGCCACAAAATGG
1810

7220_7871

HVP82
82_gen_
CGTAGTACAGCCGTTGCATTG
1811
CCCATTGTACCATTTGC
1812

1566_2325

GATAGTT

HVP82
82_gen_
GGATGTGTTGGTGTTGAAGTAGGTA
1813
TCCTGTTGGTCGTTGCCATT
1814

5821_7213

HVP82
82_gen_
GCTGCTAAGTGTATATAGTTACTCGCA
1815
CTGCTGCAAACACATA
1816

3863_4134

TTGGGATT

17.13 Summary of Example 17

The method according to the present invention described in Example 17 comprises:

1. Extraction of the viral RNA (Example 17.5) from a biological sample (Example 17.4),
2. Reverse transcription of the RNAs into cDNA with random hexamers (Example 17.6),
3. Amplification of the cDNA by multiplex PCR (Example 7) to generate a DNA sequence database.

The multiplex amplification is performed with HPV-specific primer pairs (Example 17.3)

The primers are designed specifically for each of the HPV genomes present in the database (Example 17.2).

The primers are modified to make them compatible with the high-throughput sequencing technique that is used.

4. High throughput sequencing of the DNA library and generation of “sequencing reads” (Example 17.8),
5. Aligning reads (Example 8) with the sequences of the HPV genomes present in the database (Examples 17.3 and 17.8).
6. Computing a score R (Example 17.11) whose the different possible computing are ratios described in Table 13. In this case, the ratio is defined as the ratio between the number of reads generated by at least 2 of pairs of primers described herein.

Number	Date	Country
2184368	May 2010	EP
9108312	Jun 1991	WO
9929890	Jun 1999	WO
03057914	Jul 2003	WO
2010129941	Nov 2010	WO
2011131192	Oct 2011	WO
2012116220	Aug 2012	WO

	Number	Date	Country
Parent	15319893		US
Child	17011990		US

Broad range gene and genotype papillomavirus transcriptome as a biomarker of papillomavirus-associated cancer stages

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US Referenced Citations (1)

Foreign Referenced Citations (7)

Non-Patent Literature Citations (12)

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Entry
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