LIBRARY PREPARATION METHOD AND APPLICATION

INCORPORATION BY REFERENCE

The sequence listing provided in the file entitled Sequence_listing_PCTCN2020105117.txt, which is an ASCII text file that was created on Jan. 25, 2022, and which comprises 84,565 bytes, is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the technical field of molecular biology, and particularly to a library preparation method and application.

BACKGROUND

Generally, the current sequencing and analysis of a sequence in a target region first requires a library preparation, and methods of library preparation are nothing more than capture library preparation and amplification library preparation.

The capture library preparation is an enrichment library preparation targeted at a relatively large region of a genome, such as a tens or hundreds of gene whole exon regions, while the multiplex amplification library preparation is to perform a target capture and sequencing and analysis on specific hotspot regions, or the whole exon regions of individual genes.

The method for amplification library preparation is to design corresponding specific primers according to a target region. These primers are then used to conduct a multiplex amplification on target sequences. It should be noted that these specific primers will directly carry sequencing adapters or bridging sequences, and then sequencing adapters are added thereto by a secondary PCR amplification, which is the process of a normal amplification library preparation. There are some problems in the application of the existing amplification library preparation methods. For example, the library preparation process is relatively cumbersome, requiring at least two cycles of PCR amplification and two corresponding library purifications, calling for numerous manual operation time that impose high requirements on operators, thus not conducive to popularization. Moreover, primer design and system optimization are relatively complicated; the cost of library preparation is high; and the entire library preparation process is time-consuming.

SUMMARY

Aiming at various problems existing in the amplification library preparation, the present invention provides the following technical solutions.

One purpose of the present invention is to provide a primer combination for preparing an amplicon library for detecting the variation of a target gene.

The primer combination provided by the present invention includes:

a forward outer primer F1, a forward inner primer F2, and a reverse primer R that are designed according to a target amplicon;

The forward outer primer F1 is sequentially composed of a sequencing adapter sequence 1, a barcode sequence for distinguishing different samples, and a universal sequence;

The forward inner primer F2 is sequentially composed of a universal sequence and a forward specific primer sequence of the target amplicon (a molecular tag is not required when detecting a tissue sample);

The reverse outer primer R is sequentially composed of a sequencing adapter 2 and a reverse specific primer sequence of the target amplicon.

In the above primer combination, optionally, a molecular tag is required when detecting low frequency mutations, and the forward inner primer F2 is sequentially composed of a universal sequence, a molecular tag sequence, and a forward specific primer sequence of the target amplicon.

The molecular tag sequence is composed of 6-30 bases, consisting random bases and 0-N(N is an integer ≥0) set(s) of specific bases; the specific bases are set in the random bases, for example, 1 set, 2 sets, 3 sets, or 4 sets; the specific bases in each set are composed of 1-5 bases, such as 1 base, 2 bases, 3 bases, 4 bases, or 5 bases.

The base sequence of each set is randomly selected, and the molecular tag sequence is used to distinguish different starting DNA template molecules. In a library preparation process, except for the fixed position and constant composition of the specific bases in the molecular tag sequence, the types of bases (A, T, C) of the random bases can be selected at will.

For example, in an embodiment of the present invention, the specific bases are set as 1 or 2 sets, with the sequence of ACT and/or TGA; for example, in the present embodiment, the molecular tag sequence is NNNNNACTNNNNTGA (SEQ ID NO: 13), where ACT and TGA are the specific bases, N is a random base of A, T, C, or G.

In the above primer combination, the sequencing adapter 1 and the sequencing adapter 2 are corresponding sequencing adapters selected according to different sequencing platforms.

In the above primer combination, the sequencing platform is an Illumina platform, the sequencing adapter 1 is 15, and the sequencing adapter 2 is 17;

or the sequencing platform is an Ion Torrent platform, the sequencing adapter 1 is A, and the sequencing adapter 2 is P;

or the sequencing platform is a BGI/MGI platform;

or, the nucleotide sequence of the universal sequence is shown in SEQ ID NO: 1.

Another purpose of the present invention is to provide a kit for preparing an amplicon library for detecting the variation of a target gene.

The kit provided by the present invention includes the above-mentioned primer combination.

The above kit further includes a polymerase chain reaction (PCR) amplification buffer and a DNA polymerase system.

Another purpose of the present invention is to provide any one of the following applications of the primer combination or the kit described above:

(1) an application in preparing the amplicon library for detecting the variation of the target gene;

(2) an application in detecting mutation sites or variations in a target region of a sample to be tested;

(3) an application in detecting a variation frequency of the target region of the sample to be tested.

Another purpose of the present invention is to provide a method of preparing an amplicon library for detecting a variation of a target gene.

The method provided by the present invention includes the following steps:

taking the DNA or cDNA of a sample to be tested as a template, carrying out a one-step PCR amplification using the above primer combination or the above kit to obtain an amplified product, i.e., the amplicon library of the target gene.

In the above method, the molar ratio of the forward outer primer F1, the forward inner primer F2, and the reverse primer R in an amplification system for the one-step PCR amplification is (5-20):(1-20):(5-20).

In the above method, the sample to be tested is a tissue sample, a frozen sample, a puncture sample, a formalin-fixed paraffin-embedded (FFPE) sample, blood, urine, cerebrospinal fluid, pleural fluid, or other body fluids.

The application of the above method in detecting mutation sites or variations of the target gene of the sample to be tested.

The application of the above method in detecting a variation frequency of the target gene of the sample to be tested.

The amplicon library prepared by the above method also falls within the protection scope of the present invention.

Another purpose of the present invention is to provide a method for detecting the variation of the target gene of the sample to be tested.

The method provided by the present invention includes the following steps:

1) preparing an amplicon library of the target gene by the above method;

2) evenly mixing the amplicon libraries of the target genes of all samples, and then diluting to obtain a sequencing DNA library;

3) sequencing the sequencing DNA library to obtain a sequencing result, and analyzing the variation of the target gene of the sample to be tested according to the sequencing result.

Another purpose of the present invention is to provide a method of detecting a variation frequency in a target region of a sample to be tested.

The method provided by the present invention includes the following steps:

1) preparing an amplicon library of the target gene by the above method;

2) evenly mixing the amplicon libraries of the target genes of all samples, and then diluting to obtain a sequencing DNA library;

3) sequencing the sequencing DNA library to obtain a sequencing result, and calculating the variation frequency of the target gene of the sample to be tested according to the sequencing result.

Variation frequency=number of mutation clusters/total number of effective clusters×100%.

In the above method, the sample to be tested is an in vitro tissue sample, a frozen sample, a puncture sample, an FFPE sample, blood, urine, cerebrospinal fluid, or pleural fluid.

In the above method, optionally,

the nucleotide sequence of the universal sequence is shown in SEQ ID NO: 1;

the nucleotide sequence of the sequencing adapter 1 is shown in SEQ ID NO: 2;

the nucleotide sequence of the sequencing adapter 2 is shown in SEQ ID NO: 17.

For example, when the target gene to be tested is EGFR, optionally, the corresponding forward specific primer sequence and reverse specific primer sequence are respectively shown in SEQ ID NO: 14 and SEQ ID NO: 18, or, SEQ ID NO: 15 and SEQ ID NO: 19, or, SEQ ID NO: 21 and SEQ ID NO: 24, or, SEQ ID NO: 22 and SEQ ID NO: 25;

When the target gene to be tested is ERBB2, optionally, the corresponding forward specific primer sequence and reverse specific primer sequence are respectively shown in SEQ ID NO: 16 and SEQ ID NO: 20, or, SEQ ID NO: 23 and SEQ ID NO: 26;

When the target gene to be tested is EML4, optionally, the corresponding forward specific primer sequence and reverse specific primer sequence are respectively shown in SEQ ID NO: 27 and SEQ ID NO: 31, or, SEQ ID NO: 28 and SEQ ID NO: 31;

When the target gene to be tested is LMNA, optionally, the corresponding forward specific primer sequence and reverse specific primer sequence are respectively shown in SEQ ID NO: 29 and SEQ ID NO: 32;

When the target gene to be tested is MYC, optionally, the corresponding forward specific primer sequence and reverse specific primer sequence are respectively shown in SEQ ID NO: 30 and SEQ ID NO: 33.

For example, the barcode sequences are all nucleotides with a length of 6-12 nt, no more than 3 consecutive bases, and a GC content of 40-60%;

The universal sequence 1 and the universal sequence 2 generally have a length of 16-25 nt, and a GC content of 35-65%, without consecutive bases or obvious secondary structure;

For example, the molecular tag sequence is a sequence containing 6-15 random bases; including but not limited to the above sequences; in the embodiment of the present invention, for example, the barcode sequences for distinguishing different samples are shown in SEQ ID NO: 3 to SEQ ID NO: 12;

The variation can be point mutation, deletion or insertion, or fragment fusion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the composition of primers used for a one-step rapid amplification library preparation technology.

FIG. 2 shows products obtained when amplifying a template by the rapid amplification library preparation technology.

FIG. 3 shows an Agilent 2200 result of the library prepared by a BRCA1/2 one-step primer pool.

FIG. 4 shows the homogeneity of sequencing amplicons of the library prepared by the BRCA1/2 one-step primer pool.

FIG. 5 is a schematic diagram showing the functional structure of each component of a quadruple-functional primer and a triple-functional primer.

FIG. 6 shows homogeneity results of the libraries prepared by a triple-functional component primer pool and a quadruple-functional component primer pool.

FIG. 7 shows the number of clusters (the number of molecular tag types) of one of the amplicons obtained after data analysis of the library prepared by using 30 ng cfDNA and one-step primer pool.

FIG. 8 shows the background noises at the level of 0.1‰-1‰ after sequencing the libraries prepared by the two methods.

FIG. 9 shows a result of Agilent 2200 TapeStation of the library prepared in Embodiment 2.

FIG. 10 shows a result of Agilent 2200 TapeStation of the library prepared in Embodiment 3.

FIG. 11 shows a result of Agilent 2200 TapeStation of the library prepared in Embodiment 4.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The experimental methods used in the following embodiments, unless otherwise specified, are all conventional methods.

The materials, reagents, etc. used in the following embodiments, unless otherwise specified, are commercially available.

Embodiment 1. Design and Synthesis of Primers for One-Step Amplicon Sequencing Library Preparation

I. Design of Primers for One-Step Amplicon Sequencing Library Preparation

The present invention provides an amplification library preparation method to prepare a second-generation sequencing library, and the structures of primers involved in the method are as follows (see FIG. 1):

forward outer primer F1: 5′-sequencing adapter sequence 1+Barcode sequence+universal sequence-3′;

forward inner primer F2: 5′-universal sequence+molecular tag sequence+gene forward specific primer sequence-3′;

or forward inner primer F2: 5′-universal sequence+gene forward specific primer sequence-3′ (molecular tag is required when detecting low frequency mutations, and molecular tag is not required when detecting tissue samples);

reverse primer R: 5′-sequencing adapter sequence 2+gene reverse specific primer sequence-3′.

When detecting low-frequency mutations, the structure of the forward inner primer F2 is: 5′-universal sequence+molecular tag sequence+gene forward specific primer sequence-3′.

Among them, the barcode sequence is a nucleic acid sequence that is used to distinguish different samples; a sample to be tested corresponds to a barcode sequence. The barcode sequence is 6-12 nt in length, and has no more than 3 consecutive bases, and a GC content of 40-60%, and the primer where the Barcode sequence is introduced has no obvious secondary structure, etc.

The forward outer primer F1 is used to distinguish different samples. The same sample has the same forward outer primer F1 regardless of detection sites.

The molecular tag sequence is used to mark different starting DNA template molecules (templates of different amplicons), and a starting DNA template molecule corresponds to a molecular tag sequence.

The molecular tag sequence includes random bases and at least one set of specific bases, the specific bases are set in the random bases, for example, 1 set or 2 sets; each set of specific bases is composed of 1-5 bases, for example, 3 bases or 4 bases. In a library preparation process, except for the fixed position and constant composition of the specific bases in the molecular tag sequence, the types of bases (A, T, G, C) of the random bases are randomly selected.

The starting templates of the sequencing results are classified using the molecular tag sequences, which can eliminate amplification errors and sequencing errors. In the present embodiment, two types of specific bases are used: ACT and TGA, which can be used separately or in combination.

Gene forward specific primer sequence and gene reverse specific primer sequence are primer sequences (respectively including the required forward primers and corresponding reverse primers to amplify different target regions) used to amplify specific target regions;

The universal sequence 1 is a specific nucleic acid sequence, which can be changed according to actual needs. The universal sequence 1 has a length of 16-25 nt, and a GC content of 35-65%, without consecutive bases or obvious secondary structure.

In present embodiment, the universal sequence used is GGCACCCGAGAATTCCA (SEQ ID NO: 1), with a length of 17 nt;

The sequencing adapter sequence 1 and the sequencing adapter sequence 2 are specific sequences that need to be introduced to primers during sequencing, and can specifically correspond to Ion Torrent, Illumina, or BGISEQ/MGISEQ sequencing platforms.

If the sequencing platform is the Illumina platform, the sequencing adapter sequences 1 and 2 are I5 and I7, respectively, and the adapter sequences are complementary to the primer sequences on the chip. The adapter is introduced to link a nucleic acid fragment to a vector.

If the sequencing platform is the Ion Torrent platform, the sequencing adapter sequences 1 and 2 are A and P, respectively, the A adapter is used for sequencing and complementary to the sequencing primer, and the P adapter is complementary to the sequence on the vector, so as to link a template to the vector.

If the sequencing platform is the BIISEQ/MGISEQ platform, the sequencing adapters are required for sequencing, which are specific sequences meeting the requirements of single-strand circularization, subsequent DNB preparation, and sequencing.

When the second-generation sequencing library is used in a sequencing, multiple samples will be tested simultaneously. As such, a set of forward outer primers F1 will be designed. M forward outer primers F1 correspond to M samples, and the barcode sequence in each forward outer primer F1 is different; P forward inner primers F2 and corresponding Q (generally P=Q, but there are also situations where P does not equal Q, for example, in a detection of RNA fusion genes) reverse primers R are designed according to the number P of amplicons required for the target capture region on each sample, and the structures of the molecular tags in the P forward inner primers F2 are identical.

II. Amplification Principle of One-Step Amplicon Sequencing Library

The primers design of one-step rapid amplification library preparation technology are as described above. When amplifying a template DNA/RNA, the procedure shown in FIG. 2 is followed. The forward outer primer F1 and the forward inner primer F2 share a normal universal sequence, so the forward outer primer F1 can use the forward inner primer F2 as a template to add a sequencing adapter and a sample barcode sequence to a target sequence. During amplification, the forward inner primer MIX1 (MIX1 is formed by mixing the forward inner primers F2 of multiple amplicons at a specific ratio) and the reverse primer MIX2 (MIX2 is formed by mixing the reverse primers R corresponding to multiple amplicons at a specific ratio) are used to perform the first cycle of reaction on the template to produce amplified products with F2 and R; in the second cycle of reaction, in addition to the above two PCR products, products with F2 and R sequences respectively at both ends will be further obtained; in the third cycle of reaction, a target product with the complete sequence of the complete sequencing library begins to appear, but at this time, the product has only one strand; subsequently in the fourth cycle of reaction, a double-stranded product with complete adapter sequences at two ends will be produced. Since the forward outer primer F1 has a much higher TM value and a much higher concentration than the forward inner primer F2, exponential amplifications of the complete products (that is, the two products marked with the red dashed box in the products of the fourth PCR cycle) will be realized later. Finally, the library preparation is completed after a dozen to dozens of cycles of reaction processes.

III. Establishment of Detection Method

1. One-Step Amplification

The primers synthesized in section I above were prepared as follows:

The forward outer primer F1 was dissolved in water to a primer concentration of 100 μM, and the forward inner primers F2 were respectively dissolved in water to a primer concentration of 100 μM. Subsequently, the various primers were mixed at an equimolar ratio to form the forward outer primer MIX1. The reverse primers R were respectively dissolved in water to 100 μM, and then mixed at an equimolar ratio into the reverse primer MIX2.

The genomic DNA of multiple samples to be tested was extracted.

The reagents shown in Table 1 were successively added to a 0.2 ml eight-row tube or 96-well plate (each type of nucleic acid sample was extracted according to the instruction of the specific manufacturer's kit provided in the embodiment):

Table 1 Shows the Amplification System of a Certain Sample

Reagent
Volume (μl)

KAPA HiFi PCR Kits (including but not limited
10

to the DNA polymerase)

Genomic DNA of a certain sample (generally
1-10

5-20 ng of gDNA)

Forward inner primer MIX1 (100 μM)
0.01-5

Forward outer primer F1 (100 μM)
0.01-5

Reverse primer MIX2 (100 μM)
0.01-5

DNAase-free H₂O
Replenish

water to 20

The procedure of the above PCR amplification is shown in Table 2.

Table 2 Shows the PCR Amplification Procedure

Number

Temperature
Time
of cycles

95° C.
2 m

95° C.
30 s
15-30

60° C.
90 s

72° C.
90 s

72° C.
10 m

4° C.
—

After the PCR reaction was completed, the PCR product obtained was the amplicon library.

2. Magnetic Bead Purification and Qubit Quantification

After the PCR reaction was completed, the Agencourt AMPure XP Kit (Cat. No. A63880/A63881/A63882) from Beckman Coulter Inc. was used for purification. The operation steps were as follows:

1) The Agilent court AMPure XP Kit was taken out 30 min in advance, fully vortex and put aside at room temperature.

2) After the PCR reaction, the magnetic beads were fully vortexed again, 24 μl of magnetic beads were added to the system, blow repeatedly more than 5 times or vortex fully, and put aside at room temperature for 5 min.

3) The Eppendorf (EP) tubes were transferred to a magnetic stand and put aside for 5 min until the solution was clear, by using a pipette to carefully remove the supernatant without contacting the magnetic beads.

4) 100 μl of freshly prepared 80% ethanol solution was added to each tube, the EP tubes were slowly rotated for 2 cycles on the magnetic stand, followed by putting aside for 5 min and discarding the supernatant.

5) The step 4 was repeated one more time.

6) The EP tubes were opened and put aside at room temperature to allow a complete liquid volatilization until surfaces of the magnetic beads became matte. The magnetic beads should not be dried excessively.

7) The EP tubes were removed from the magnetic stand, 30 μl of PCR-grade purified water was added, followed by vortex to mix well, and putting aside at room temperature for 10 min.

8) The EP tubes in the previous step were placed on the magnetic stand for 2 min or until the solution was clear, followed by using a pipette to carefully absorb the supernatant on the side away from the magnet without contacting the magnetic beads.

A purified amplicon library was obtained.

The purified amplicon library was subjected to a DNA library concentration determination and an Agilent 2200 TapeStation Systems detection using Qubit 2.0.

3. Sequencing and Result Analysis

The purified amplicon libraries of multiple samples were mixed at an equal concentration, and then diluted to 100 PM to obtain a DNA library for amplicon sequencing. Sequencing was performed (sequenator used was Ion GeneStudio™ S5 Plus System, Thermofisher, A38195), after data processing and analysis (S5 Torrent Server), the mutations and mutation frequency of a tested sample were obtained.

The calculation method of the variation frequency of the library with molecular tags was as follows:

Since the original template was subjected to molecular marking during the library amplification process, the calculation method of the mutation frequency was as follows:

In the sequencing results, DNA molecules with the same kind of molecular tags were defined as a cluster, and DNA molecules with the same kind of molecular tags were amplified products of an initial DNA template, that is, a series of DNA molecules obtained by amplification using the same original template;

Whether mutations occurred in the cluster or not was confirmed. If the proportion of a specific type of bases in a certain position in the cluster was greater than or equal to 80%, the cluster was recorded as an effective cluster. If the number of mutant DNA molecules with molecular tags in the effective cluster accounted for greater than or equal to 80%, it was recorded as a mutation cluster;

Variation frequency=number of mutation clusters/total number of effective clusters×100%.

Notes: It is statistically significant only when the number of DNA molecules in the same cluster (a sequence sequenced) in the sequencing results is ≥2.

Embodiment 2. Preparation and Sequencing of One-Step Amplicon Sequencing Library

1. Design of Primers for One-Step Amplicon Sequencing Library Preparation

The detection region of this experiment contained three amplicons (EGFR L858R, 19del and insertion mutations of ERBB2);

The test samples included two frozen lung cancer tissue samples (sample 1, sample 2), four lung cancer FFPE (formalin fixed paraffin-embedded tissue samples) samples (sample 3, sample 4, sample 5, sample 6), and two white blood cell samples from healthy subjects (sample 7, sample 8). The mutations of the above eight samples were already known.

The primers (eight Barcode sequences were used in the present embodiment) shown in Table 3 were designed according to the three amplicons (EGFR L858R, 19del and insertion mutations of ERBB2):

Table 3 Shows the Primer Sequences of EGFR L858R, 19Del and Insertion Mutations of ERBB2

Primer

Required primer
Amplicon
sequence

Forward
Sequencing

CCATCTCATC

outer
adapter

CCTGCGTGTC

primer F1
sequence 1

TCCGACTCAG

(SEQ ID

NO: 2)

Barcode

TCCTCGAATC

sequence

(SEQ ID

(ten

NO: 3)

listed)

TAGGTGGTTC

(SEQ ID

NO: 4)

TCTAACGGAC

(SEQ ID

NO 5)

TTGGAGTGTC

(SEQ ID

NO: 6)

TCTAGAGGTC

(SEQ ID

NO: 7)

TCTGGATGAC

(SEQ ID

NO: 8)

TCTATTCGTC

(SEQ ID

NO: 9)

AGGCAATTGC

(SEQ ID

NO: 10)

TTAGTCGGAC

(SEQ ID

NO: 11)

CAGATCCATC

(SEQ ID

NO: 12)

Universal

GGCACCCGAG

sequence

AATTCCA

(SEQ ID

NO: 1)

Forward
Universal

GGCACCCGAG

inner
sequence

AATTCCA

sequence

(SEQ ID

F2

NO: 1)

Gene
EGFR
CAGGAACGTA

forward
L858R
CTGGTGAAAA

specific

CAC

primer

(SEQ ID

sequence

NO: 14)

EGFR
CTTCCTTCTC

19del
TCTCTGTCAT

AGGGA

(SEQ ID

NO: 15)

ERBB2
CTCCCATACC

CTCTCAGCGT

A (SEQ ID

NO: 16)

Reverse
Sequencing

CCTCTCTATG

primer R
adapter

GGCAGTCGGT

sequence 2

GAT (SEQ ID

NO: 17)

Gene
EGFR
GAAAATGCTG

reverse
L858R
GCTGACCTAA

specific

AGC

primer

(SEQ ID

sequence

NO: 18)

EGFR
AGCAAAGCAG

19del
AAACTCACAT

CGA

(SEQ ID

NO: 19)

ERBB2
AGCCATAGGG

CATAAGCTGT

G

(SEQ ID

NO: 20)

The sequencing adapter is suitable for the Ion GeneStudio™ S5 Plus System sequencing platform.

II. One-Step Amplicon Sequencing Library

Nucleic acid extraction and purification kit (DNA extraction from FFPE samples: GeneRead DNA FFPE kit, Qiagen, 180134; DNA extraction from frozen tissue samples: QIAamp DNA Mini Kit 250, QIAGEN, 51306).

1. One-Step Amplification

The PCR product was obtained according to step 1 in section III of Embodiment 1.

The amplification system is shown in Table 4.

Table 4 Shows the Amplification System

Reagent
Volume (μl)

KAPA HiFi PCR Kits (including but not limited
10

to the DNA polymerase)

Genomic DNA of a certain sample
5-20

Forward inner primer MIX1 (100 μM)
1

Forward outer primer F1 (100 μM)
0.5

Reverse primer MIX2 (100 μM)
0.5

DNAase-free H₂O
Replenish

water to 20

Table 5 Shows the Amplification Procedure

Number

Temperature
Time
of cycles

95° C.
2 m

95° C.
30 s
18

60° C.
90 s

72° C.
90 s

72° C.
10 m

4° C.
—

2. Magnetic Bead Purification and Qubit Quantification

Same steps were performed as those in step 2 of section III of Embodiment 1.

The PCR product was purified and recovered by the magnetic bead (Agencourt AMPure XP, Beckman Coulter, A63880), and DNA library concentration determination and Agilent 2200 TapeStation Systems detection were conducted using Qubit 2.0.

The result of the Agilent 2200 TapeStation Systems detection is shown in FIG. 9.

3. Sequencing and Result Analysis

The PCR products of all samples were mixed at an equal concentration and diluted to 100 pM to obtain a DNA library for sequencing.

The sequencing results are shown in Table 6:

Table 6 Shows the Results of Sequencing

Method of the present invention
63 gene detection

Variation

Variation

Sample No.
Variation type
frequency
Variation type
frequency

Frozen sample 1
EGFR: L858R
13.7%
EGFR: L858R
17.8%

Frozen sample 2
EGFR: p.E746_A75
8.1%
EGFR: p.E746_A75
7.3%

0delELREA

0delELREA

FFPE sample 1
EGFR: L858R
33.5%
EGFR: L858R
31.9%

FFPE sample 2
EGFR: L858R
21.0%
EGFR: L858R
19.2%

FFPE sample 3
EGFR: p.K745_E74
23.8%
EGFR: p.K745_E74
23.5%

9delKELRE

9delKELRE

FFPE sample 4
ERBB2: p.A775_G
17.2%
ERBB2: p.A775_G
17.1%

776insYVMA

776insYVMA

White blood cell
None
0
None
0

sample 1 from

healthy subject

White blood cell
None
0
None
0

sample 2 from

healthy subject

EGFR: p.E746_A750delELREA indicates a deletion of the 746^th-750^thamino acids ELREA (E: Glu glutamic acid; L: Leu leucine; R: Arg arginine; E: Glu glutamate; A: Ala alanine) of the EGFR gene, which is a kind of EGFR 19del;

EGFR: p.K745_E749delKELRE indicates a deletion of the 745^th-749^thamino acids KELRE (K: Lys lysine; E: Glu glutamic acid; L: Leu leucine; R: Arg arginine; E: Glu glutamic acid) of the EGFR gene, which is a kind of EGFR 19del;

ERBB2: p.A775_G776insYVMA indicates an insertion of YVMA (Y: Tyr Tyrosine; V: Val Valine; M: Met Methionine; A: Ala alanine) between the 775^thalanine (A) and the 776^thglycine (G) of the ERBB2 gene, corresponding to ERBB2 in Table 3.

The 63 gene detection product is a product of tumor liquid biopsy of Genetron Health (Beijing) Co., Ltd. It targets all solid tumor patients and applies high-throughput and high-precision second-generation sequencing technology to comprehensively detect mutations of 63 gene loci closely related to tumor-targeted therapy and occurrence and development (including mutation analysis of 58 genes, rearrangement analysis of 10 genes, and CNV detection of 7 genes), covering the target region with a sequencing depth of 20,000×, and reaching a detection sensitivity of 0.1%, which provides comprehensive and high-value reference information for precise medication, molecular typing, and curative effect and recurrence monitoring.

The above results show that the library prepared by the method of the present invention, when used for sequencing, leads to the variation information of tested tissue samples including point mutations, deletion mutations and insertion mutations consistent with that obtained by the known 63 gene detection.

Embodiment 3. Preparation and Sequencing of One-Step Amplicon Sequencing Library

The samples in this experiment were plasma samples from lung cancer patients, including plasma samples from four different patients and two healthy subjects (the variations of the samples were already known), cfDNA was extracted using the kit (MagMAX™ Cell-Free DNA Isolation Kit, Applied Biosystems™, A29319), and the library was prepared using a primer pool with molecular tags containing EGFR L858R, 19del and insertion mutations of ERBB2.

I. Design of Primers for One-Step Amplicon Sequencing Library Preparation

The primers (forward outer primers were identical, others were different, and six barcode sequences were used in the present embodiment) shown in Table 7 were designed according to three amplicons (EGR L858R, 19del and insertion mutations of ERBB2):

Table 7 Shows the Primer Sequences of EGR L858R, 19Del and Insertion Mutations of ERBB2

Primer

Require primer
Amplicon
sequence

Forward
Universal

GGCACCCGA

inner
sequence 1

GAATTCCA

primer

(SEQ ID

F2

NO: 1)

Molecular

NNNNNACT

tag

NNNNTGA

sequence

(SEQ ID

NO: 13),

where the

Bold

Letters

are

specific

bases.

Gene
EGFR
GGAGGACC

forward
L858R
GTCGCTTG

specific

(SEQ ID

primer

NO: 21)

sequence

Gene
EGFR
GTGAGAAA

forward
19del
GTTAAAAT

specific

TCCCGTC

primer

(SEQ ID

sequence

NO: 22)

Sequencing

adapter
ERBB2
CCCATACC

sequence 2

CTCTCAGC

GT

(SEQ ID

NO: 23)

CCTCTCTA

TGGGCAGT

CGGTGAT

(SEQ ID

NO: 17)

Reverse
Gene
EGFR
CTTCTGCA

primer R
reverse
L858R
TGGTATTC

specific

TTTCTCTT

primer

CC

sequence

(SEQ ID

NO: 24)

Gene
EGFR
CACACAGC

reverse
19del
AAAGCAGA

specific

AAC

primer

(SEQ ID

sequence

NO: 25)

ERBB2
CCAGAAGG

CGGGAGAC

ATATG

(SEQ ID

NO: 26)

II. One-Step Amplicon Sequencing Library

1. One-Step Amplification

The PCR product was obtained according to step 1 in section III of Embodiment 1.

Table 8 Shows the Amplification System

Reagent
Volume (μl)

KAPA HiFi PCR Kits (including but not limited
10

to the DNA polymerase)

Genomic DNA of a certain sample
5-20

Forward inner primer MIX1 (100 μM)
0.5

Forward outer primer F1 (100 μM)
1

Reverse primer MIX2 (100 μM)
1

DNAase-free H₂O
Replenish

water to 20

Table 9 Shows the Amplification Procedure

Number

Temperature
Time
of cycles

95° C.
2 m

95° C.
30 s
2

65° C.
30 s

62° C.
30 s

59° C.
30 s

72° C.
30 s

95° C.
30 s
16

60° C.
30 s

72° C.
30 s

72° C.
10 m

4° C.
—

2. Magnetic Bead Purification and Qubit Quantification

Same steps were performed as those in step 2 of section III of Embodiment 1.

The PCR product was purified and recovered by the magnetic bead (Agencourt AMPure XP, Beckman Coulter, A63880), and detected by Qubit 2.0 and Agilent 2200 TapeStation Systems.

The result of the Agilent 2200 TapeStation Systems is shown in FIG. 10.

3. Sequencing and Result Analysis

The PCR products of all samples were mixed at an equal concentration and diluted to 100 μM to obtain a DNA library for amplicon sequencing.

The sequencing results are shown in Table 10:

Table 10 Shows the Detection Results of Four Tissue Samples and Two Samples from Healthy Subjects

Method of the

present invention
63 gene detection

Variation

Variation

Sample No.
Variation type
frequency
Variation type
frequency

Patient 1
EGFR: L858R
0.57%
EGFR: L858R
0.72%

Patient 2
EGFR: L858R
0.21%
EGFR: L858R
0.18%

Patient 3
EGFR: p.E746_A
0.80%
EGFR: p.E746_A750
0.93%

750delELREA

delELREA

Patient 4
ERBB2: p.A775_
0.38%
ERBB2: p.A775_G7
0.35%

G776insYVMA

76insYVMA

Healthy subject 1
None
0
None
0

Healthy subject 2
None
0
None
0

The library prepared by the method of the present invention, when used for sequencing, leads to variation information of tested plasma cfDNA samples including point mutations, deletion mutations and insertion mutations consistent with that obtained by the known 63 gene detection. The amount of ctDNA extracted from the patient 1 sample is large. After the patient 1 sample is diluted by 5 times, the detection of L858R with a frequency of 4.6‰ is still obtained (after deduplicating the data Reads: mutation cluster=2; total cluster at the locus=4380).

Embodiment 4. Preparation and Sequencing of One-Step Amplicon Sequencing Library

The samples in this experiment were fine-needle aspiration (FNA) puncture samples of 3 thyroid cancer patients with gene fusion (gene fusion information was already known) and FNA puncture samples of 2 patients with benign thyroid nodules. RNA samples were extracted using MagMAX™ FFPE DNA/RNA Ultra Kit (Applied Biosystems™, A31881) according to the manufacturer's instruction, and then reverse transcription was conducted using SuperScript™ VILO™ MasterMix (Invitrogen™, 11755050) according to the manufacturer's kit instruction.

I. Design of Primers for One-Step Amplicon Sequencing Library Preparation

The primers (forward outer primers were identical to those in Table 3, others were different, and five barcode sequences were used in the present embodiment) shown in Table 11 were designed according to gene fusion: the primers for detecting gene fusion were designed before and after the breakpoint, and there was no fixed forward and reverse primer matching; the forward and reverse primers designed for the fusion breakpoint were shown as below, ALK_20 and ELM4_6/EML4_13 were combined separately to detect two ALK-EML4 fusion forms.

Table 11 Shows the Primers of Gene Fusion

Primer

Require primer
Amplicon
sequence

Forward
Universal

GGCACCCGA

inner
sequence 1

GAATTCCA

primer F2

(SEQ ID

NO: 1)

Gene
EML4_6_
ACTGCAGAC

forward

AAGCATAAA

specific

GATGTCA

primer

(SEQ ID

NO: 27)

sequence
EML4J3
ACTACTGTA

GAGCCCACA

CCTG

(SEQ ID

NO: 28)

LMNA
CTGAGAACA

GGCTGCAGA

CC

(SEQ ID

NO: 29)

MYC
CCTGGTGCT

CCATGAGGA

GA

(SEQ ID

NO: 30)

Reverse
Sequencing

CCTCTCTAT

primer R
adapter

GGGCAGTCG

sequence 2

GTGAT

(SEQ ID

NO: 17)

Gene
ALK20
CTCAGCTTG

reverse

TACTCAGGG

specific

CTC

primer

(SEQ ID

sequence

NO: 31)

LMNA
ACTCACGCT

GCTTCCCAT

T

(SEQ ID

NO: 32)

MYC
GTGATCCAG

ACTCTGACC

TTTTGC

(SEQ ID

NO: 33)

II. One-Step Amplicon Sequencing Library

1. One-Step Amplification

The PCR product was obtained according to step 1 in section III of Embodiment 1.

Table 12 Shows the Amplification System

Reagent
Volume (μl)

Platinum multiplex PCR Master Mix
15

Thy RNA Fusion Panel
2

Barcode (50 μM)
1

cDNA
≤12

ddH₂O
Replenish to 30

Table 13 Shows the Amplification Procedure

Number

Temperature
Time
of cycles

95° C.
2 min

95° C.
30 s
18

60° C.
90 s

72° C.
90 s

72° C.
10 min

4° C.
∞

2. Magnetic Bead Purification and Qubit Quantification

Same steps were performed as those in step 2 of section III of Embodiment 1.

The PCR product was purified and recovered by the magnetic bead (Agencourt AMPure XP, Beckman Coulter, A63880), and detected by Qubit 2.0 and Agilent 2200 TapeStation Systems.

The result of the Agilent 2200 TapeStation Systems detection is shown in FIG. 11.

3. Sequencing and Result Analysis

The PCR products of all samples were mixed at an equal concentration and diluted to 100 μM to obtain a DNA library for amplicon sequencing.

The sequencing results are shown in Table 14:

Table 14 Shows the Comparison of Detection Results of Gene Fusion

Sample No.
Method of the present invention
63 gene detection

Patient 1
EML4-ALK-V3a (E6a A20)
EML4-ALK-V3a (E6a A20)

Patient 2
EML4-ALK-V3b (E6b A20)
EML4-ALK-V3b (E6b A20)

Patient 3
EML4-ALK-V1 (E13 A20)
EML4-ALK-V1 (E13 A20)

Healthy subject 1
None
None

Healthy subject 2
None
None

EML4-ALK-V3a (E6a A20) corresponds to EML4_6 and ALK_20 in Table 11;

EML4-ALK-V1 (E13 A20) corresponds to EML4_13 and ALK_20 in Table 11.

The 63 gene detection product used Agilent's customized probes to perform capture library preparation. The product has been used for detecting thousands of clinical plasma samples, and the performance of the product is stable.

The library prepared by the method of the present invention, when used for sequencing, leads to fusion mutation forms of tested samples consistent with the mutation information of samples obtained by the known 63 gene detection.

The foregoing embodiments are only used to illustrate the present invention. The structure, connection mode, and manufacturing process of each component can be changed. Any equivalent transformation and improvement based on the technical solution of the present invention should not be excluded from the protection scope of the present invention.

Embodiment 5. BRCA1/2 One-Step Primer Pool

I. Design of Primers for One-Step Amplicon Sequencing Library Preparation

The forward outer primers were the same as those in Table 3, others were different, and the barcodes were determined according to the number of samples in the library preparation.

The forward inner primer F2: universal sequence+forward specific primer sequence

The reverse primer R: sequencing adapter 2+reverse specific primer sequence

Table 15 Shows the BRCA1/2 Primer Set

Universal sequence
GGCACCCGAGAATTCCA

(SEQ ID NO: 1)

Sequencing adapter 2
CCTCTCTATGGG

CAGTCGGTGAT

(SEQ ID NO: 17)

Forward
P1_B1_F1
cacctacctg

specific

ataccccaga

primer

tccc

sequence

(SEQ ID NO: 34)

P1_B1_F2
ccctggagtc

gattgattag

agccta

(SEQ ID NO: 35)

P1_B1_F3
cagttccagt

agtcctactt

tgacact

(SEQ ID NO: 36)

P1_B1_F4
tcatcattca

cccttggcac

agtaa

(SEQ ID NO: 37)

P1_B1_F5
taagccttca

tccggagagt

gta

(SEQ ID NO: 38)

P1_B1_F6
cttttataac

tagattttcc

ttctctccat

tcc

(SEQ ID NO: 39)

P1_B1_F7
ggtccaaagc

gagcaagaga

atcc

(SEQ ID NO: 40)

P1_B1_F8
cgcctggcct

gaatgcctta

aa

(SEQ ID NO: 41)

P1_B1_F9
aagagcacgt

tcttctgctg

tatg

(SEQ ID NO: 42)

P1_B1_F10
gaaatatttt

ctaggaattg

cgggagga

(SEQ ID NO: 43)

P1_B1_F11
atccagattg

atcttgggag

tgtaaaaa

(SEQ ID NO: 44)

P1_B1_F12
tgtgtgctag

aggtaactca

tgataatgg

(SEQ ID NO: 45)

P1_B1_F13
agaaagggtc

aacaaaagaa

tgtccat

(SEQ ID NO: 46)

P1_B1_F14
tgaaagttcc

ccaattgaaa

gttgcag

(SEQ ID NO: 47)

P1_B1_F15
gaactttgta

attcaacatt

catcgttgtg

t

(SEQ ID NO: 48)

P1_B1_F16
ttagatgata

ggtggtacat

gcacagtt

(SEQ ID NO: 49)

P1_B1_F17
taccagtaaa

aataaagaac

caggagtgg

(SEQ ID NO: 50)

P1_B1_F18
aacctgaatt

atcactatca

gaacaaagca

(SEQ ID NO: 51)

P1_B1_F19
tgaacagtac

ccgttccctt

ga

(SEQ ID NO: 52)

P1_B1_F20
ccttgaggac

ctgcgaaatc

cag

(SEQ ID NO: 53)

P1_B1_F21
atggaaagct

tctcaaagta

tttcattttc

t

(SEQ ID NO: 54)

Pl_B1_F22
tgcagcgttt

atagtctgct

tttacatc

(SEQ ID NO: 55)

P1_B1_F23
gaacgggctt

ggaagaaaat

aatcaag

(SEQ ID NO: 56)

P1_B1_F24
ttctgctagc

ttgttttctt

cacagt

(SEQ ID NO: 57)

P1_B1_F25
aaacaatata

ccttctcagt

ctactaggca

t

(SEQ ID NO: 58)

P1_B1_F26
gctgttttta

gcaaaagcgt

ccaga

(SEQ ID NO: 59)

P1_B1_F27
tcagataact

tagaacagcc

tatgggaag

(SEQ ID NO: 60)

P1_B1_F28
gggccaaaat

tgaatgctat

gcttagat

(SEQ ID NO: 61)

P1_B1_F29
gagcacaatt

agccgtaata

acattagaga

a

(SEQ ID NO: 62)

P1_B1_F30
ctggactcat

tactccaaat

aaacatgga

(SEQ ID NO: 63)

P1_B1_F31
agtctaatat

caagcctgta

cagacagtt

(SEQ ID NO: 64)

P1_B1_F32
ttgcagaata

cattcaaggt

ttcaaagc

(SEQ ID NO: 65)

P1_B1_F33
aaataaatgt

gtgagtcagt

gtgcag

(SEQ ID NO: 66)

P1_B1_F34
ataatgctga

agaccccaaa

gatctc

(SEQ ID NO: 67)

P1_B1_F35
agccaaatga

acagacaagt

aaaagaca

(SEQ ID NO: 68)

P1_B1_F36
tgcaaattga

tagttgttct

agcagtgaa

(SEQ ID NO: 69)

P1_B1_F37
gcagcagtat

aagcaatatg

gaactcgaa

(SEQ ID NO: 70)

P1_B1_F38
cggagcagaa

tggtcaagtg

atgaata

(SEQ ID NO: 71)

P1_B1_F39
aagagcgtcc

cctcacaaat

aaatt

(SEQ ID NO: 72)

P1_B1_F40
tgaaagagtt

cactccaaat

cagtagaga

(SEQ ID NO: 73)

P1_B1_F41
aggttctgat

gactcacatg

atggg

(SEQ ID NO: 74)

P1_B1_F42
cccctgtgtg

agagaaaaga

atggaataa

(SEQ ID NO: 75)

P1_B1_F43
aaggctgaat

tctgtaataa

aagcaaaca

(SEQ ID NO: 76)

P1_B1_F44
cagggtagtt

ctgtttcaaa

cttgcat

(SEQ ID NO: 77)

P1_B1_F45
ttgtatattt

tcagctgctt

gtgaattttc

t

(SEQ ID NO: 78)

P1_B1_F46
tgacagttct

gcatacatgt

aactagtgt

(SEQ ID NO: 79)

P1_B1_F47
ctagttgaat

atctgttttt

caacaagtac

atttt

(SEQ ID NO: 80)

P1_B1_F48
agcggataca

acctcaaaag

acg

(SEQ ID NO: 81)

P1_B1_F49
gtgtcaagtt

tctcttcagg

aggaaaag

(SEQ ID NO: 82)

P1_B1_F50
aaaggaaaat

aactctcctg

aacatctaaa

aga

(SEQ ID NO: 83)

P1_B1_F51
ttgttgaaga

gctattgaaa

atcatttgtg

c

(SEQ ID NO: 84)

P1_B1_F52
attatagagg

ttttctactg

ttgctgcat

(SEQ ID NO: 85)

P1_B1_F53
ggcagttgtg

agattatctt

ttcatggc

(SEQ ID NO: 86)

P1_B1_F54
ctctgagaaa

gaatgaaatg

gagttgg

(SEQ ID NO: 87)

P1_B2_Fl
aaacaaattt

tccagcgctt

ctg

(SEQ ID NO: 88)

P1_B2_F2
ggtaaaaatg

cctattggat

ccaaaga

(SEQ ID NO: 89)

P1_B2_F3
tggtttgaag

aactttcttc

agaagc

(SEQ ID NO: 90)

P1_B2_F4
tcttcttaca

actccctata

cattctcat

(SEQ ID NO: 91)

P1_B2_F5
agtgaaaact

aaaatggatc

aagcagat

(SEQ ID NO: 92)

P1_B2_F6
aaactagttt

ttgccagttt

tttaaaataa

cc

(SEQ ID NO: 93)

P1_B2_F7
tttttacccc

cagtggtatg

tg

(SEQ ID NO: 94)

P1_B2_F8
tgtacctagc

attctgcctc

ata

(SEQ ID NO: 95)

P1_B2_F9
ggatcctgat

atgtcttggt

caagtt

(SEQ ID NO: 96)

P1_B2_F10
tgaagaagca

tctgaaactg

tatttcc

(SEQ ID NO: 97)

P1_B2_Fll
ggactactac

tatatgtgca

ttgagagttt

(SEQ ID NO: 98)

P1_B2_F12
gaaaacacaa

atcaaagaga

agctgc

(SEQ ID NO: 99)

P1_B2_F13
tggcttataa

aatattaatg

tgcttctgtt

t

(SEQ ID NO: 100)

P1_B2_F14
aatctacaaa

aagtaagaac

tagcaagac

(SEQ ID NO: 101)

P1_B2_F15
aagtgacaaa

atctccaagg

aagttgt

(SEQ ID NO: 102)

P1_B2_F16
gaattctttg

ccacgtattt

ctagc

(SEQ ID NO: 103)

P1_B2_F17
ggcttcttca

tttcagggta

tcaaaa

(SEQ ID NO: 104)

P1_B2_F18
aatacatact

gtttgctcac

agaagga

(SEQ ID NO: 105)

P1_B2_F19
accgaaagac

caaaaatcag

aactaattaa

(SEQ ID NO: 106)

P1_B2_F20
tcacagaatg

attctgaaga

accaac

(SEQ ID NO: 107)

P1_B2_F21
attaccccag

aagctgattc

tctg

(SEQ ID NO: 108)

P1_B2_F22
tatatgatca

tgaaaatgcc

agcactc

(SEQ ID NO: 109)

P1_B2_F23
ttcccatgga

aaagaatcaa

gatgtat

(SEQ ID NO: 110)

P1_B2_F24
actgtcaatc

cagactctga

agaact

(SEQ ID NO: 111)

P1_B2_F25
caggtgataa

acaagcaacc

caag

(SEQ ID NO: 112)

P1_B2_F26
caaatgggca

ggactcttag

g

(SEQ ID NO: 113)

P1_B2_F27
tggcattaga

taatcaaaag

aaactgag

(SEQ ID NO: 114)

P1_B2_F28
gaatcaggaa

gtcagtttga

atttactca

(SEQ ID NO: 115)

P1_B2_F29
gcctgttgaa

aaatgactgt

aacaaaa

(SEQ ID NO: 116)

P1_B2_F30
gtgaggaaac

ttctgcagag

g

(SEQ ID NO: 117)

P1_B2_F31
tgaagataac

aaatatactg

ctgccag

(SEQ ID NO: 118)

P1_B2_F32
aggagggaaa

cactcagatt

aaagaag

(SEQ ID NO: 119)

P1_B2_F33
tttcagactg

caagtgggaa

aaatat

(SEQ ID NO: 120)

P1_B2_F34
ccagttggta

ctggaaatca

actagt

(SEQ ID NO: 121)

P1_B2_F35
aaaagagcaa

ggtactagtg

aaatcac

(SEQ ID NO: 122)

P1_B2_F36
aaaaaccttg

tttctattga

gactgtg

(SEQ ID NO: 123)

P1_B2_F37
aattcagcct

tagcttttta

cacaagt

(SEQ ID NO: 124)

P1_B2_F38
tgacaaaaat

catctctccg

aaaaaca

(SEQ ID NO: 125)

P1_B2_F39
gccagtattg

aagaatgttg

aagatcaaa

(SEQ ID NO: 126)

P1_B2_F40
aataattttg

aggtagggcc

acct

(SEQ ID NO: 127)

P1_B2_F41
tcataactct

ctagataatg

atgaatgtag

c

(SEQ ID NO: 128)

P1_B2_F42
gtatagggaa

gcttcataag

tcagtct

(SEQ ID NO: 129)

P1_B2_F43
agaagatagt

accaagcaag

tcttttc

(SEQ ID NO: 130)

P1_B2_F44
tagtacagca

agtggaaagc

aagt

(SEQ ID NO: 131)

P1_B2_F45
ctcagaaatg

gaaaaaacct

gcagtaa

(SEQ ID NO: 132)

P1_B2_F46
caggcttcac

ctaaaaacgt

aaaaat

(SEQ ID NO: 133)

P1_B2_F47
catgccacac

attctctttt

tacatg

(SEQ ID NO: 134)

P1_B2_F48
atataccata

cctatagagg

gagaacagat

at

(SEQ ID NO: 135)

P1_B2_F49
acattcactg

aaaattgtaa

agcctataat

t

(SEQ ID NO: 136)

P1_B2_F50
atatattttc

tccccattgc

agcaca

(SEQ ID NO: 137)

P1_B2_F51
aggacatcca

ttttatcaag

tttctgc

(SEQ ID NO: 138)

P1_B2_F52
tggctctgat

gatagtaaaa

ataagattaa

tg

(SEQ ID NO: 139)

P1_B2_F53
ggttgtgctt

tttaaatttc

aattttattt

ttgc

(SEQ ID NO: 140)

P1_B2_F54
gttccctctg

cgtgttctca

ta

(SEQ ID NO: 141)

P1_B2_F55
gctgtatacg

tatggcgttt

ctaaaca

(SEQ ID NO: 142)

P1_B2_F56
agttgtagtt

gttgaattca

gtatcatcc

(SEQ ID NO: 143)

P1_B2_F57
tgtgcctttc

ctaaggaatt

tgctaat

(SEQ ID NO: 144)

P1_B2_F58
aaaagataat

ggaaagggat

gacacag

(SEQ ID NO: 145)

P1_B2_F59
ctgttaaggc

ccagttagat

cct

(SEQ ID NO: 146)

P1_B2_F60
aggcagttct

agaagaatga

aaactct

(SEQ ID NO: 147)

P1_B2_F61
tagacctttt

cctctgccct

tatc

(SEQ ID NO: 148)

P1_B2_F62
cacattatta

cagtggatgg

agaagac

(SEQ ID NO: 149)

P1_B2_F63
cttctttggg

tgttttatgc

ttggt

(SEQ ID NO: 150)

P1_B2_F64
gcagagcttt

atgaagcagt

gaag

(SEQ ID NO: 151)

P1_B2_F65
tcttaaatgg

tcacagggtt

atttcag

(SEQ ID NO: 152)

P1_B2_F66
ggatgtcaca

accgtgtg

(SEQ ID NO: 153)

P1_B2_F67
ttccattgca

tctttctcat

ctttct

(SEQ ID NO: 154)

Reverse
P1_B1_R1
atatttagta

specific

gccaggacag

primer

tagaagg

sequence

(SEQ ID NO: 155)

P1_B1_R2
gtagagtgct

acactgtcca

ac

(SEQ ID NO: 156)

P1_B1_R3
ataaaccaaa

cccatgcaaa

agga

(SEQ ID NO: 157)

P1_B1_R4
cccttacaga

tggagtcttt

tgg

(SEQ ID NO: 158)

P1_B1_R5
gatgaaagct

ccttcaccac

aga

(SEQ ID NO: 159)

P1_B1_R6
ccactatgta

agacaaaggc

tgg

(SEQ ID NO: 160)

P1_B1_R7
aagaacctgt

gtgaaagtat

ctagca

(SEQ ID NO: 161)

P1_B1_R8
gtggtttctt

ccattgacca

cat

(SEQ ID NO: 162)

P1_B1_R9
gcattgatgg

aaggaagcaa

atac

(SEQ ID NO: 163)

P1_B1_R10
aaagaccttt

tggtaactca

gactca

(SEQ ID NO: 164)

P1_B1_R11
aaatatttca

gtgtccgttc

acacaca

(SEQ ID NO: 165)

P1_B1_R12
gcagatgcaa

ggtattctgt

aaag

(SEQ ID NO: 166)

P1_B1_R13
acctacataa

aactctttcc

agaatgttg

(SEQ ID NO: 167)

P1_B1_R14
ccctttctgt

tgaagctgtc

aatt

(SEQ ID NO: 168)

P1_B1_R15
agatggtatg

ttgccaacac

ga

(SEQ ID NO: 169)

P1_B1_R16
gatgtttccg

tcaaatcgtg

tg

(SEQ ID NO: 170)

P1_B1_R17
agcaataaaa

gtgtataaat

gcctgtatg

(SEQ ID NO: 171)

P1_B1_R18
gtagaactat

ctgcagacac

ctcaaa

(SEQ ID NO: 172)

P1_B1_R19
ccagaaccac

catctttcag

taattt

(SEQ ID NO: 173)

P1_B1_R20
atcataaaat

gttggagcta

ggtcct

(SEQ ID NO: 174)

P1_B1_R21
tatgatggaa

gggtagctgt

tagaag

(SEQ ID NO: 175)

P1_B1_R22
ggttaaaatg

tcactctgag

aggatag

(SEQ ID NO: 176)

P1_B1_R23
ggaaatttgt

aaaatgtgct

ccccaa

(SEQ ID NO: 177)

P1_B1_R24
aattccttgt

cactcagacc

aact

(SEQ ID NO: 178)

P1_B1_R25
actaaggtga

tgttcctgag

atg

(SEQ ID NO: 179)

P1_B1_R26
ggaagcaggg

aagctcttca

t

(SEQ ID NO: 180)

P1_B1_R27
actttcctta

atgtcatttt

cagcaaaac

(SEQ ID NO: 181)

P1_B1_R28
cagtctgaac

tacttcttca

tattcttgc

(SEQ ID NO: 182)

P1_B1_R29
ctagttctgc

ttgaatgttt

tcatcac

(SEQ ID NO: 183)

P1_B1_R30
tggaatgttc

tcatttccca

tttctct

(SEQ ID NO: 184)

P1_B1_R31
gtttcgttgc

ctctgaactg

aga

(SEQ ID NO: 185)

P1_B1_R32
ccttgatttt

cttccttttg

ttcacattc

(SEQ ID NO: 186)

P1_B1_R33
tttctatgct

tgtttcccga

ctg

(SEQ ID NO: 187)

P1_B1_R34
cctagagtgc

taacttccag

taac

(SEQ ID NO: 188)

P1_B1_R35
cttggaaggc

taggattgac

aaattc

(SEQ ID NO: 189)

P1_B1_R36
ttgttactct

tcttggctcc

agtt

(SEQ ID NO: 190)

P1_B1_R37
ttaggtgggc

ttagatttct

actgac

(SEQ ID NO: 191)

P1_B1_R38
tgcttatagg

ttcagctttc

gtttt

(SEQ ID NO: 192)

P1_B1_R39
tccgtttggt

tagttccctg

atttat

(SEQ ID NO: 193)

P1_B1_R40
gtattatctg

tggctcagta

acaaatg

(SEQ ID NO: 194)

P1_B1_R41
ttaaagcctc

atgaggatca

ctg

(SEQ ID NO: 195)

P1_B1_R42
agttcatcac

ttctggaaaa

ccact

(SEQ ID NO: 196)

P1_B1_R43
gggatcagca

ttcagatcta

cctttt

(SEQ ID NO: 197)

P1_B1_R44
ttcagccttt

tctacattca

ttctgtc

(SEQ ID NO: 198)

P1_B1_R45
taccctgata

cttttctgga

tgcc

(SEQ ID NO: 199)

P1_B1_R46
gaatccaaac

tgatttcatc

cctgg

(SEQ ID NO: 200)

P1_B1_R47
ccagcttcat

agacaaaggt

tctc

(SEQ ID NO: 201)

P1_B1_R48
agctgcctac

cacaaataca

aattat

(SEQ ID NO: 202)

P1_B1_R49
cagagttctc

acagttccaa

ggtta

(SEQ ID NO: 203)

P1_B1_R50
gaagaagaag

aaaacaaatg

gttttaccaa

(SEQ ID NO: 204)

P1_B1_R51
atcaccacgt

catagaaagt

aattgtg

(SEQ ID NO: 205)

P1_B1_R52
tcaacaagtt

gactaaatct

cgtactttc

(SEQ ID NO: 206)

P1_B1_R53
cattcttaca

taaaggacac

tgtgaag

(SEQ ID NO: 207)

P1_B1_R54
ctctgagaaa

gaatgaaatg

gagttgg

(SEQ ID NO: 208)

P1_B2_R1
ggcattttta

cctacgatat

tcctccaatg

(SEQ ID NO: 209)

P1_B2_R2
tgtgacgtac

tgggttttta

gcaag

(SEQ ID NO: 210)

P1_B2_R3
gagtcagccc

ttgctctttg

aat

(SEQ ID NO: 211)

P1_B2_R4
ttcactgtgc

gaagactttt

atgtcta

(SEQ ID NO: 212)

P1_B2_R5
ggctcttagc

caaaatatta

gcataaaaat

cag

(SEQ ID NO: 213)

P1_B2_R6
taaaaagcat

tgifittaat

catacctgac

tt

(SEQ ID NO: 214)

P1_B2_R7
aggtacagat

ttgtaaatct

cagggcaa

(SEQ ID NO: 215)

P1_B2_R8
acctcagctc

ctagactttc

agaaatatg

(SEQ ID NO: 216)

P1_B2_R9
gatgacaatt

atcaacctca

tctgctctt

(SEQ ID NO: 217)

P1_B2_R10
aggtttagag

actttctcaa

aggcttagat

(SEQ ID NO: 218)

P1_B2_R11
tgtgttttca

ctgtctgtca

cagaag

(SEQ ID NO: 219)

P1_B2_R12
cgagatcacg

ggtgacagag

c

(SEQ ID NO: 220)

P1_B2_R13
aaaaactatc

ttcttcagag

gtatctacaa

ct

(SEQ ID NO: 221)

P1_B2_R14
gggcttctga

tttgctacat

ttgaatct

(SEQ ID NO: 222)

P1_B2_R15
taggtctttt

tctgaaatat

tttggtcaca

tg

(SEQ ID NO: 223)

P1_B2_R16
cagatattgc

ctgctttact

gcaagaa

(SEQ ID NO: 224)

P1_B2_R17
atgtatttcc

agtccacttt

cagagg

(SEQ ID NO: 225)

P1_B2_R18
tttgttttat

tttcaaagtg

gatattaaac

ct

(SEQ ID NO: 226)

P1_B2_R19
acagaaggaa

tcgtcatcta

taaaactata

tgt

(SEQ ID NO: 227)

P1_B2_R20
ctgtagtttt

tccttattac

attttgcttc

tt

(SEQ ID NO: 228)

P1_B2_R21
ctgggattga

aagtcagtat

cactgtatt

(SEQ ID NO: 229)

P1_B2_R22
tgttaccttt

gagcttgtct

gacattttg

(SEQ ID NO: 230)

P1_B2_R23
tttggattac

tcttagattt

gtgttttggt

tg

(SEQ ID NO: 231)

P1_B2_R24
catggtagag

ttcttgaaaa

tgggttc

(SEQ ID NO: 232)

P1_B2_R25
ggtattttat

ctatattcaa

ggagatgtcc

gatt

(SEQ ID NO: 233)

P1_B2_R26
acaatttcaa

cacaagctaa

actagtagga

t

(SEQ ID NO: 234)

P1_B2_R27
tgccttttgg

ctaggtgtta

aattatgg

(SEQ ID NO: 235)

P1_B2_R28
tgtctacctg

accaatcgat

ggg

(SEQ ID NO: 236)

P1_B2_R29
cagctttttg

cagagcttca

gtaga

(SEQ ID NO: 237)

P1_B2_R30
ttcaacaaaa

gtgccagtag

tcatttc

(SEQ ID NO: 238)

P1_B2_R31
tggccagata

atttaagaca

tatgttgtgc

(SEQ ID NO: 239)

P1_B2_R32
tgctccgttt

tagtagcagt

taactgt

(SEQ ID NO: 240)

P1_B2_R33
tgtctgtttc

ctcataactt

agaatgtcca

t

(SEQ ID NO: 241)

P1_B2_R34
ttttcacttt

gtccaaagat

tcctttgc

(SEQ ID NO: 242)

P1_B2_R35
gagaattctg

catttcttta

cactttggg

(SEQ ID NO: 243)

P1_B2_R36
gggactgatt

tgtgtaacaa

gttgcag

(SEQ ID NO: 244)

P1_B2_R37
ttcatacaaa

taatttccta

cataatctgc

agt

(SEQ ID NO: 245)

P1_B2_R38
tcaatactgg

ctcaatacca

gaatcaagt

(SEQ ID NO: 246)

P1_B2_R39
ttttgcaggg

tgaagagcta

gtc

(SEQ ID NO: 247)

P1_B2_R40
caacctgcca

taattttcgt

ttggc

(SEQ ID NO: 248)

P1_B2_R41
tgaagtttcc

aaactaacat

cacaaggtg

(SEQ ID NO: 249)

P1_B2_R42
tatttcagaa

aacacttgtc

ttgcgtt

(SEQ ID NO: 250)

P1_B2_R43
taccacatta

tatgaaaagc

ctttttggg

(SEQ ID NO: 251)

P1_B2_R44
gggtttctct

tatcaacacg

aggaagt

(SEQ ID NO: 252)

P1_B2_R45
cccaaaacat

gaatgttctc

aacaagtg

(SEQ ID NO: 253)

P1_B2_R46
tctgtcagtt

catcatcttc

cataaaagc

(SEQ ID NO: 254)

P1_B2_R47
tagcatacca

agtctactga

ataaacactt

t

(SEQ ID NO: 255)

P1_B2_R48
atgaaatatt

tattttagga

gaaccctcaa

(SEQ ID NO: 256)

P1_B2_R49
acaggtaatc

ggctctaaag

aaacatg

(SEQ ID NO: 257)

P1_B2_R50
tgcttgaaga

tttttccaaa

gtcagatgt

(SEQ ID NO: 258)

P1_B2_R51
tgttttgctt

ttgtctgttt

tcctccaa

(SEQ ID NO: 259)

P1_B2_R52
aaggcaaaaa

ttcatcacac

aaattgtca

(SEQ ID NO: 260)

P1_B2_R53
tcagagagat

tcgaggcaga

gtg

(SEQ ID NO: 261)

P1_B2_R54
cattcctgca

ctaatgtgtt

cattct

(SEQ ID NO: 262)

P1_B2_R55
atcattggag

ggtatgagcc

atcc

(SEQ ID NO: 263)

P1_B2_R56
tgccagtttc

catatgatcc

atctatagt

(SEQ ID NO: 264)

P1_B2_R57
cagaaacctt

aaccatactg

ccgtatatg

(SEQ ID NO: 265)

P1_B2_R58
ggccactttt

tgggtatctg

cacta

(SEQ ID NO: 266)

P1_B2_R59
cttcaagagg

tgtacaggca

tcag

(SEQ ID NO: 267)

P1_B2_R60
gggtcaggaa

agaatccaag

tttggtata

(SEQ ID NO: 268)

P1_B2_R61
gaaactccat

ctcaaacaaa

caaacaaatt

aat

(SEQ ID NO: 269)

P1_B2_R62
tectectgaa

ttttagtgaa

taaggcttct

(SEQ ID NO: 270)

P1_B2_R63
tgcaaagcac

gaacttgctg

t

(SEQ ID NO: 271)

P1_B2_R64
tgtgatggcc

agagagtcta

aaacag

(SEQ ID NO: 272)

P1_B2_R65
gtgacatccc

ttgataaacc

ttgttcc

(SEQ ID NO: 273)

P1_B2_R66
tagtagtgga

ttttgcttct

ctgatataaa

ct

(SEQ ID NO: 274)

P1_B2_R67
ttttttgtcg

ctgctaactg

tatgtta

(SEQ ID NO: 275)

II. One-Step Amplicon Sequencing Library

1. One-Step Amplification

The PCR product was obtained using 0.5 pg of gDNA of white blood cell in plasma from healthy subject as a starting sample according to step 1 in section III of Embodiment 1.

2. Magnetic Bead Purification and Qubit Quantification

Same steps were performed as those in step 2 of section III of Embodiment 1.

The PCR product was purified and recovered by the magnetic bead (Agencourt AMPure XP, Beckman Coulter, A63880), and detected by Qubit 2.0 and Agilent 2200 TapeStation Systems.

The result of the Agilent 2200 TapeStation Systems detection is shown in FIG. 3. The prepared library is highly specific, and does not have non-specific amplification products or primer dimers. The prepared library has high quality and is suitable for sequencing.

3. Sequencing and Result Analysis

The PCR products of all samples were mixed at an equal concentration and diluted to 100 μM to obtain a DNA library for amplicon sequencing.

The sequencing results are shown in FIG. 4. The sequencing analysis results show that the 121 amplicons of the BRCA1/2 detection library has a good homogeneity, indicating the advantages of the one-step library preparation technology of the present invention in terms of amplicon homogeneity, and ensuring an effective output of data.

Comparison of three primers used in the method of the comparative example and the method of the present invention and four primers in the prior art

I. Design of Primers for One-Step Amplicon Sequencing Library Preparation

The structures of the 3 primers and the 4 primers designed are shown in FIG. 5.

The Present Invention:

The 3 primers of the present invention were designed according to the design principle of Embodiment 1:

The forward outer primers were the same as those in Table 3, specifically, there were 67 barcode sequences; the universal sequences were the same as those in Table 15, and the forward specific gene sequences were P1_B2_F1 to P1_B2_F67 in Table 15;

The sequencing adapter 2 was the same as that in Table 15, and the reverse specific primer sequences were P1_B2_R1 to P1_B2_R67 in Table 15;

Control:

The 4 primers were designed according to the following principles in the prior art:

Design Principles:

Barcode primer F1: sequencing adapter 1+barcode sequence+universal sequence 1;

Forward inner primer F2: universal sequence 1+molecular tag+specific base sequence+forward specific primer sequence;

Reverse outer primer R1: sequencing adapter 2+universal sequence 2;

Reverse inner primer R2: universal sequence 2+reverse specific primer sequence;

The sequencing adapter 1+barcode sequence described above are shown in Table 3.

The rest sequences are shown in Table 16 below:

Table 16 Shows the Control Primer Sequences

Universal sequence 1
GGCACCCGAGAATTC

CA

(SEQ ID

NO: 1)

Universal sequence 2
CCACTACGCCTCCGC

TTT

(SEQ ID

NO: 410)

Sequencing adapter 2
CCTCTCTATGGGCAG

TCGGTGAT

(SEQ ID

NO: 17)

Forward
P1_B2_F1
aaacaaattttccag

specific

cgcttctg

primer

(SEQ ID

sequence

NO: 276)

P1_B2_F2
ggtaaaaatgcctat

tggatccaaaga

(SEQ ID

NO: 277)

P1_B2_F3
tggtttgaagaactt

tcttcagaagc

(SEQ ID

NO: 278)

P1_B2_F4
tcttcttacaactcc

ctatacattctcat

(SEQ ID

NO: 279)

P1_B2_F5
agtgaaaactaaaat

ggatcaagcagat

(SEQ ID

NO: 280)

P1_B2_F6
aaactagtttttgcc

agttttttaaaataa

cc

(SEQ ID

NO: 281)

P1_B2_F7
tttttacccccagtg

gtatgtg

(SEQ ID

NO: 282)

P1_B2_F8
tgtacctagcattct

gcctcata

(SEQ ID

NO: 283)

P1_B2_F9
ggatcctgatatgtc

ttggtcaagtt

(SEQ ID

NO: 284)

P1_B2_F10
tgaagaagcatctga

aactgtatttcc

(SEQ ID

NO: 285)

P1_B2_F11
ggactactactatat

gtgcattgagagttt

(SEQ ID

NO: 286)

P1_B2_F12
gaaaacacaaatcaa

agagaagctgc

(SEQ ID

NO: 287)

P1_B2_F13
tggcttataaaatat

taatgtgcttctgtt

t

(SEQ ID

NO: 288)

P1_B2_F14
aatctacaaaaagta

agaactagcaagac

(SEQ ID

NO: 289)

P1_B2_F15
aagtgacaaaatctc

caaggaagttgt

(SEQ ID

NO: 290)

P1_B2_F16
gaattctttgccacg

tatttctagc

(SEQ ID

NO: 291)

P1_B2_F17
ggcttcttcatttca

gggtatcaaaa

(SEQ ID

NO: 292)

P1_B2_F18
aatacatactgtttg

ctcacagaagga

(SEQ ID

NO: 293)

P1_B2_F19
accgaaagaccaaaa

atcagaactaattaa

(SEQ ID

NO: 294)

P1_B2_F20
tcacagaatgattct

gaagaaccaac

(SEQ ID

NO: 295)

P1_B2_F21
attaccccagaagct

gattctctg

(SEQ ID

NO: 296)

P1_B2_F22
tatatgatcatgaaa

atgccagcactc

(SEQ ID

NO: 297)

P1_B2_F23
ttcccatggaaaaga

atcaagatgtat

(SEQ ID

NO: 298)

P1_B2_F24
actgtcaatccagac

tctgaagaact

(SEQ ID

NO: 299)

P1_B2_F25
caggtgataaacaag

caacccaag

(SEQ ID

NO: 300)

P1_B2_F26
caaatgggcaggact

cttagg

(SEQ ID

NO: 301)

P1_B2_F27
tggcattagataatc

aaaagaaactgag

(SEQ ID

NO: 302)

P1_B2_F28
gaatcaggaagtcag

tttgaatttactca

(SEQ ID

NO: 303)

P1_B2_F29
gcctgttgaaaaatg

actgtaacaaaa

(SEQ ID

NO: 304)

P1_B2_F30
gtgaggaaacttctg

cagagg

(SEQ ID

NO: 305)

P1_B2_F31
tgaagataacaaata

tactgctgccag

(SEQ ID

NO: 306)

P1_B2_F32
aggagggaaacactc

agattaaagaag

(SEQ ID

NO: 307)

P1_B2_F33
tttcagactgcaagt

gggaaaaatat

(SEQ ID

NO: 308)

P1_B2_F34
ccagttggtactgga

aatcaactagt

(SEQ ID

NO: 309)

P1_B2_F35
aaaagagcaaggtac

tagtgaaatcac

(SEQ ID

NO: 310)

P1_B2_F36
aaaaaccttgtttct

attgagactgtg

(SEQ ID

NO: 311)

P1_B2_F37
aattcagccttagct

ttttacacaagt

(SEQ ID

NO: 312)

P1_B2_F38
tgacaaaaatcatct

ctccgaaaaaca

(SEQ ID

NO: 313)

P1_B2_F39
gccagtattgaagaa

tgttgaagatcaaa

(SEQ ID

NO: 314)

P1_B2_F40
aataattttgaggta

gggccacct

(SEQ ID

NO: 315)

P1_B2_F41
tcataactctctaga

taatgatgaatgtag

c

(SEQ ID

NO: 316)

P1_B2_F42
gtatagggaagcttc

ataagtcagtct

(SEQ ID

NO: 317)

P1_B2_F43
agaagatagtaccaa

gcaagtcttttc

(SEQ ID

NO: 318)

P1_B2_F44
tagtacagcaagtgg

aaagcaagt

(SEQ ID

NO: 319)

P1_B2_F45
ctcagaaatggaaaa

aacctgcagtaa

(SEQ ID

NO: 320)

P1_B2_F46
caggcttcacctaaa

aacgtaaaaat

(SEQ ID

NO: 321)

P1_B2_F47
catgccacacattct

ctttttacatg

(SEQ ID

NO: 322)

P1_B2_F48
atataccatacctat

agagggagaacagat

at

(SEQ ID

NO: 323)

P1_B2_F49
acattcactgaaaat

tgtaaagcctataat

t

(SEQ ID

NO: 324)

P1_B2_F50
atatattttctcccc

attgcagcaca

(SEQ ID

NO: 325)

P1_B2_F51
aggacatccatttta

tcaagtttctgc

(SEQ ID

NO: 326)

P1_B2_F52
tggctctgatgatag

taaaaataagattaa

tg

(SEQ ID

NO: 327)

P1_B2_F53
ggttgtgctttttaa

atttcaattttattt

ttgc

(SEQ ID

NO: 328)

P1_B2_F54
gttccctctgcgtgt

tctcata

(SEQ ID

NO: 329)

P1_B2_F55
gctgtatacgtatgg

cgtttctaaaca

(SEQ ID

NO: 330)

P1_B2_F56
agttgtagttgttga

attcagtatcatcc

(SEQ ID

NO: 331)

P1_B2_F57
tgtgcctttcctaag

gaatttgctaat

(SEQ ID

NO: 332)

P1_B2_F58
aaaagataatggaaa

gggatgacacag

(SEQ ID

NO: 333)

P1_B2_F59
ctgttaaggcccagt

tagatcct

(SEQ ID

NO: 334)

P1_B2_F60
aggcagttctagaag

aatgaaaactct

(SEQ ID

NO: 335)

P1_B2_F61
tagaccttttcctct

gcccttatc

(SEQ ID

NO: 336)

P1_B2_F62
cacattattacagtg

gatggagaagac

(SEQ ID

NO: 337)

P1_B2_F63
cttctttgggtgttt

tatgcttggt

(SEQ ID

NO: 338)

P1_B2_F64
gcagagctttatgaa

gcagtgaag

(SEQ ID

NO: 339)

P1_B2_F65
tcttaaatggtcaca

gggttatttcag

(SEQ ID

NO: 340)

P1_B2_F66
ggatgtcacaaccgt

gtg

(SEQ ID

NO: 341)

P1_B2_F67
ttccattgcatcttt

ctcatctttct

(SEQ ID

NO: 342)

Reverse
P1_B2_R1
ggcatttttacctac

specific

gatattcctccaatg

primer

(SEQ ID

sequence

NO: 343)

P1_B2_R2
tgtgacgtactgggt

ttttagcaag

(SEQ ID

NO: 344)

P1_B2_R3
gagtcagcccttgct

ctttgaat

(SEQ ID

NO: 345)

P1_B2_R4
ttcactgtgcgaaga

cttttatgtcta

(SEQ ID

NO: 346)

P1_B2_R5
ggctcttagccaaaa

tattagcataaaaat

cag

(SEQ ID

NO: 347)

P1_B2_R6
taaaaagcattgttt

ttaatcatacctgac

tt

(SEQ ID

NO: 348)

P1_B2_R7
aggtacagatttgta

aatctcagggcaa

(SEQ ID

NO: 349)

P1_B2_R8
acctcagctcctaga

ctttcagaaatatg

(SEQ ID

NO: 350)

P1_B2_R9
gatgacaattatcaa

cctcatctgctctt

(SEQ ID

NO: 351)

P1_B2_R10
aggtttagagacttt

ctcaaaggcttagat

(SEQ ID

NO: 352)

P1_B2_R11
tgtgttttcactgtc

tgtcacagaag

(SEQ ID

NO: 353)

P1_B2_R12
cgagatcacgggtga

cagagc

(SEQ ID

NO: 354)

P1_B2_R13
aaaaactatcttctt

cagaggtatctacaa

ct

(SEQ ID

NO: 355)

P1_B2_R14
gggcttctgatttgc

tacatttgaatct

(SEQ ID

NO: 356)

P1_B2_R15
taggtctttttctga

aatattttggtcaca

tg

(SEQ ID

NO: 357)

P1_B2_R16
cagatattgcctgct

ttactgcaagaa

(SEQ ID

NO: 358)

P1_B2_R17
atgtatttccagtcc

actttcagagg

(SEQ ID

NO: 359)

P1_B2_R18
tttgttttctttttc

aaagtggatattaaa

cct

(SEQ ID

NO: 360)

P1_B2_R19
acagaaggaatcgtc

atctataaaactata

tgt

(SEQ ID

NO: 361)

P1_B2_R20
ctgtagtttttcctt

attacattttgcttc

tt

(SEQ ID

NO: 362)

P1_B2_R21
ctgggattgaaagtc

agtatcactgtatt

(SEQ ID

NO: 363)

P1_B2_R22
tgttacctttgagct

tgtctgacattttg

(SEQ ID

NO: 364)

P1_B2_R23
tttggattactctta

gatttgtgttttggt

tg

(SEQ ID

NO: 365)

P1_B2_R24
catggtagagttctt

gaaaatgggttc

(SEQ ID

NO: 366)

P1_B2_R25
ggtattttatctata

ttcaaggagatgtcc

gatt

(SEQ ID

NO: 367)

P1_B2_R26
acaatttcaacacaa

gctaaactagtagga

t

(SEQ ID

NO: 368)

P1_B2_R27
tgccttttggctagg

tgttaaattatgg

(SEQ ID

NO: 369)

P1_B2_R28
tgtctacctgaccaa

tcgatggg

(SEQ ID

NO: 370)

P1_B2_R29
cagctttttgcagag

cttcagtaga

(SEQ ID

NO: 371)

P1_B2_R30
ttcaacaaaagtgcc

agtagtcatttc

(SEQ ID

NO: 372)

P1_B2_R31
tggccagataattta

agacatatgttgtgc

(SEQ ID

NO: 373)

P1_B2_R32
tgctccgttttagta

gcagttaactgt

(SEQ ID

NO: 374)

P1_B2_R33
tgtctgtttcctcat

aacttagaatgtcca

t

(SEQ ID

NO: 375)

P1_B2_R34
ttttcactttgtcca

aagattcctttgc

(SEQ ID

NO: 376)

P1_B2_R35
gagaattctgcattt

ctttacactttggg

(SEQ ID

NO: 377)

P1_B2_R36
gggactgatttgtgt

aacaagttgcag

(SEQ ID

NO: 378)

P1_B2_R37
ttcatacaaataatt

tcctacataatctgc

agt

(SEQ ID

NO: 379)

P1_B2_R38
tcaatactggctcaa

taccagaatcaagt

(SEQ ID

NO: 380)

P1_B2_R39
ttttgcagggtgaag

agctagtc

(SEQ ID

NO: 381)

P1_B2_R40
caacctgccataatt

ttcgtttggc

(SEQ ID

NO: 382)

P1_B2_R41
tgaagtttccaaact

aacatcacaaggtg

(SEQ ID

NO: 383)

P1_B2_R42
tatttcagaaaacac

ttgtcttgcgtt

(SEQ ID

NO: 384)

P1_B2_R43
taccacattatatga

aaagcctttttggg

(SEQ ID

NO: 385)

P1_B2_R44
gggtttctcttatca

acacgaggaagt

(SEQ ID

NO: 386)

P1_B2_R45
cccaaaacatgaatg

ttctcaacaagtg

(SEQ ID

NO: 387

P1_B2_R46
tctgtcagttcatca

tcttccataaaagc

(SEQ ID

NO: 388)

P1_B2_R47
tagcataccaagtct

actgaataaacactt

t

(SEQ ID

NO:

389)

P1_B2_R48
atgaaatatttcttt

ttaggagaaccctca

a

(SEQ ID

NO: 390)

P1_B2_R49
acaggtaatcggctc

taaagaaacatg

(SEQ ID

NO: 391)

P1_B2_R50
tgcttgaagattttt

ccaaagtcagatgt

(SEQ ID

NO: 392)

P1_B2_R51
tgttttgcttttgtc

tgttttcctccaa

(SEQ ID

NO: 393)

P1_B2_R52
aaggcaaaaattcat

cacacaaattgtca

(SEQ ID

NO: 394)

P1_B2_R53
tcagagagattcgag

gcagagtg

(SEQ ID

NO: 395)

P1_B2_R54
cattcctgcactaat

gtgttcattct

(SEQ ID

NO: 396)

P1_B2_R55
atcattggagggtat

gagccatcc

(SEQ ID

NO: 397)

P1_B2_R56
tgccagtttccatat

gatccatctatagt

(SEQ ID

NO: 398)

P1_B2_R57
cagaaaccttaacca

tactgccgtatatg

(SEQ ID

NO: 399)

P1_B2_R58
ggccactttttgggt

atctgcacta

(SEQ ID

NO: 400)

P1_B2_R59
cttcaagaggtgtac

aggcatcag

(SEQ ID

NO: 401)

P1_B2_R60
gggtcaggaaagaat

ccaagtttggtata

(SEQ ID

NO: 402)

P1_B2_R61
gaaactccatctcaa

acaaacaaacaaatt

aat (SEQ ID

NO: 403)

P1_B2_R62
tcctcctgaatttta

gtgaataaggcttct

(SEQ ID

NO: 404)

P1_B2_R63
tgcaaagcacgaact

tgctgt

(SEQ ID

NO: 405)

P1_B2_R64
tgtgatggccagaga

gtctaaaacag

(SEQ ID

NO: 406)

P1_B2_R65
gtgacatcccttgat

aaaccttgttcc

(SEQ ID

NO: 407)

P1_B2_R66
tagtagtggattttg

cttctctgatataaa

ct

(SEQ ID

NO: 408)

P1_B2_R67
ttttttgtcgctgct

aactgtatgtta

(SEQ ID

NO: 409)

II. One-Step Amplicon Sequencing Library

The method was the same as that in step 2 of Embodiment 2.

The sequencing results are analyzed as follows:

1. The Homogeneity Results of the Libraries Prepared by Triple-Functional Component Primer Pool and Quadruple-Functional Component Primer Pool

The homogeneity of the amplicons library is a very important indicator of the quality of the library. Good homogeneity of the library indicates a higher coverage of the target region of the library, and a better detection accuracy of the panel covering region. For this purpose, under the premise of ensuring the intact functional structure of the primer, the primer design of the amplicon is improved. The improved primer structure is optimized and simplified from the original F1+F2+R1+R2 (quadruple-functional primer components) to F1+F2+R (triple-functional primer components). This design will increase the stability of the reaction system and ensure the homogeneity of amplicons in the library.

Amplifications were respectively carried out on the primer set of the present invention and the control primer set using the same white blood cell DNA sample as a template.

The results are shown in FIG. 6. When the ratio of the specific primer is not adjusted, the comparison between the homogeneity of amplicons in the library prepared by triple-functional primer components and the homogeneity of amplicons in the library prepared by quadruple-functional primer components of the library of 67 amplicons (67 pairs of amplicons of BRCA2 selected from the 121 pairs of primers in Embodiment 5) indicates that the triple-functional component primer has significant advantages in the homogeneity of the library.

2. 30 ng of cfDNA was Used in a Library Preparation by One-Step Primer Pool, and the Number of Molecular Tag Types/the Number of Clusters of One of the Amplicons was Obtained after Data Analysis

Amplifications were respectively carried out on the primer set of the present invention and the control primer set using the same cfDNA sample as a template.

The results are shown in FIG. 7. Compared with the quadruple-functional component primer, the triple-functional component primer has better capture efficiency of original template than the quadruple-functional component primer, which makes the ultra-low frequency detection more sensitive and stable. The figure below is an amplicon randomly selected in the triple-functional component primer method, and after library preparation, the data information after adding tags to the original template is obtained. The higher template capture efficiency allows the triple-functional component primer method to reach a lower detection limit of mutation frequency.

3. Background Noise at the Level of 0.1‰-1‰ of the Libraries Prepared by Two Methods and Subjected to Sequencing (Same as 2)

Amplifications were respectively carried out on the primer set of the present invention and the control primer set using the same cfDNA sample as a template.

The results are shown in FIG. 8. Compared with the amplicon library preparation method of the quadruple-functional component primer, using the triple-functional component primer effectively improves the capture efficiency of the template, reduces the non-specific amplification of the library, and decreases the number of cycles of the library amplification. At the same time, through the comparison of two amplicon library preparation methods, it is found that under the use of high-fidelity DNA polymerase, the triple-functional component primer is better in terms of the background noise of sequencing data at the level of 5‰. The lower background noise enables the triple-functional primer component method to be more accurate in detecting a relatively low frequency mutation.

Good amplification homogeneity, high capture efficiency of original template molecules, high-fidelity DNA polymerase, ultra-low background noise, and the introduction of molecular tags eventually facilitate the triple-functional primer component to achieve an effective detection of ultra-low frequency mutation at the level of 3‰. The primer structure of this library preparation method has been fully optimized, and the performance of this library preparation method is much better than that of the traditional library preparation method for low-frequency mutation detection.

The comparison results of the one-step rapid library preparation method of the present invention, the ordinary amplification library preparation method and the capture library preparation method are shown in Table 17 and Table 18.

Table 17 Shows the Comparison of the One-Step Rapid Library Preparation Method, the Ordinary Amplification Library Preparation Method, and the Capture Library Preparation Method

One-step rapid library
Ordinary amplification
Capture library

preparation
library preparation
preparation

Sample required
Very little
little
Much (100-500 ng)

Sample capture
Very high
high
Relatively high

efficiency

operation
<5 min
Relatively complicated
Very complicated

Library preparation
<1.5 h
8 h
2 d

time

Contamination risk
Extremely low risk of
Potential contamination
Potential contamination

cross-contamination
risk
risk

Laboratory
Low
Relatively high
High

requirement

Quantification
Qubit quantification
qPCR quantification
qPCR quantification

method

Flexibility
Good
Good
Poor (difficult to

increase or decrease

capture region)

Capture region
Moderate
Moderate
Very large

Library preparation
Very low
Relatively high
Very high

cost

Operator requirement
Low
Relatively high
Very high

Table 18 Shows the Comparison Results of the Proportion of Target Fragments in the Library of the Present Method and the Control Method

Proportion of main peak of
Proportion of main peak of

Sample No
RIN
the one-step library
the control library

LAAAFST1
3.2
50.66%
30.28%

PC949TQ2
2.9
100%
57.48%

PA970TQ1
2.8
100%
56.71%

LAAAF0T1
2.8
100%
73.34%

LAAAFPT1
2.7
84.86%
59.94%

PD010TQ1
2.3
80.17%
69.85%

PC916TQ1
2.2
100%
82.54%

PC980TQ1
2
100%
52.30%

PC977TP1
1.7
100%
51.83%

LAAAEVT1
1.7
100%
38.01%

After the amplicon library is prepared, there may be amplification products of target fragments, primer dimers or multimers, and fragment products of non-specific amplification in the system. A high proportion of the amplification products of target fragments becomes an extremely important indicator for evaluating the quality of the amplicon library. Table 18 shows the present method has great advantages in terms of the proportion of target fragments of the library as compared to the control method.

INDUSTRIAL APPLICATION

In order to solve the current difficulties in library preparation, the present invention has developed the one-step rapid amplification library preparation method. Compared with the traditional capture method, the amplification library preparation method has the following advantages (FIG. 1). The library preparation method is simple and rapid, has a low requirement for operators, and can achieve the library preparation by only a normal PCR operation for corresponding reaction time. Since the quality and purity of the library prepared by this method are very high, only a simple cycle of magnetic bead purification and Qubit quantification are required before being used in a normal sequencing. The one-step library preparation technology can be applied to all second-generation platforms including IonTorrent, illumina and BGI/MGI platforms. Based on the library preparation method, the present invention has developed detection products targeted at SNP, Ins/Del, CNV and methylation of DNA, as well as detection products for gene fusion and expression of RNA samples.

The present invention has the following merits because of adopting the above technical solutions:

1. Little sample consumption and high utilization rate. The capture efficiency of the original template molecules in the sample is high, and thus a relatively low amount of starting templates is required. When performing germline mutation detection, even just a pg-level amount of starting templates is required. When performing low frequency mutation detection of cfDNA, a limited amount of starting templates can achieve a higher template capture efficiency, thereby achieving an effective capture of trace ctDNA molecules, realizing a lower detection limit and a higher sensitivity;

2. Ultra-low detection limit. The unique primer design, supporting PCR reaction system, reaction conditions, and subsequent information analysis and noise reduction system ultimately result in the lowest mutation detection limit of 3‰, making it possible to realize an accurate detection of ultra-early stage and trace amounts of ctDNA sample mutations;

3. Good homogeneity of library. The innovative primer structure design and supporting reaction system result in the optimal homogeneity of amplicons in the library. When conducting a multiplex amplification, the different structural characteristics of the sequences of various amplicons and the different amplification efficiencies of various primers will eventually result in a huge difference in the abundance of amplicons in the library. How to balance the difference in the abundance of amplicons is a key indicator to evaluate the quality of the library. The components of the triple-functional primer used in the present method have obvious advantages over the components of the quadruple-functional primer. Specifically, the cooperation of primer composition and reaction system ensures that the method can control differential amplifications of amplicons at a reduced number of cycles, and then a method like universal primer amplification is used. Since there is no competition between R1 and R2 in the following figure, a stable low differential amplification is achieved in subsequent cycles;

4. High repeatability. The components of the quadruple-functional primer will increase the uncertainty of the reaction system and reaction conditions, and are more sensitive to sample quality, reaction system and external environmental influences. While the components of the triple-functional primer have been improved in this aspect, and the simpler components result in a better system stability, and a higher repeatability and accuracy of sample detection;

5. Easy operation and time saving. The traditional capture library preparation technology has cumbersome operations and long procedures. The entire library preparation process takes nearly 48 h and imposes high requirements on operators. The ordinary amplification library preparation method requires at least two cycles of PCR and two cycles of purification, including subsequent QPCR quantification. The entire library preparation process requires at least one working day. The present invention only involves one-step PCR reaction and corresponding product purification steps, and the entire library preparation process can be completed within 1.5 h, thereby simplifying the library preparation operation process and saving time of the library preparation (the library preparation can be completed within 1.5 h, and the entire process from the library preparation to the completion of sequencing and to the completion of the bioinformatic analysis can be controlled within 22 h);

6. Able to detect multiple gene mutation types. Starting from a DNA sample, SNP, SNV, Ins/Del, methylation, gene or exon level copy number variation, and chromosome arm level copy number variation can be detected. In addition, after adding molecular tags to primers, mutations at the level of as low as 1‰ can be further detected. Starting with a RNA sample, the expression of specific genes, the fusion of specific genes, etc. can be detected;

7. Multiple sample types. The starting sample can be fresh tissue samples, frozen samples, puncture samples, FFPE samples and other tissue sample types. Meanwhile, isolated cfDNA or CTC in blood, urine, cerebrospinal fluid, and pleural fluid can also be detected. After DNA or RNA is extracted from normal samples, library preparation can be conducted by one-step rapid amplification library preparation method;

8. Effective elimination of cross-contamination between samples. The barcode sequences that distinguish different samples are added at the beginning of PCR, and the simplification of the operation process and steps effectively eliminates possible cross-contaminations during the library preparation process, especially when detecting low frequency mutations, cross-contamination between samples is extremely prone to determining as a false positive mutation;

9. Reduced cost of library preparation. Compared with the traditional capture technology, the cost required for library preparation using the present method is greatly reduced. The capture probes used in the traditional capture library preparation are expensive, and the reagents and consumables involved in the lengthy experimental process also increase the cost of capture library preparation. In contrast, the one-step library preparation process requires a greatly reduced amount of reagents and consumables, and the cost of library preparation is much lower than that of the traditional capture library preparation method. At the same time, compared with the one-step rapid amplification library preparation method, at least one cycle of additional PCR and purification and the QPCR quantitation of the library in the normal amplification library preparation method will also greatly increase the cost of library preparation. Compared with the components of the prior quadruple-functional primer, the components of the triple-functional primer lead to low consumption of total primer and each component of primer, thus having a lower cost advantage;

10. Space saving. Since this method requires only one cycle of PCR, the laboratory requires only 3 rooms (sample extraction, PCR amplification room, library purification and sequencing), which saves space as compared to the conventional library preparation where 4 rooms (sample extraction, PCR1, PCR2, and library purification and sequencing) are required.

Flexible and simple library preparation method, allowing detection of multiple mutation types, and extremely high detection sensitivity are the biggest features of the present invention.

LIBRARY PREPARATION METHOD AND APPLICATION

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