Patent Application
20220230706
The disclosure is based on the priority of Japanese patent application No. 2019-102716 (filed on May 31, 2019), and the entire contents of the same application are incorporated by reference into the application. The disclosure relates to an information processing apparatus, an information processing method and an information processing program. Particularly, the disclosure relates to an information processing apparatus, an information processing method and an information processing program for DNA profiling.
DNA profiling using microsatellites has been performed. The microsatellites include repeat sequences, thus a phenomenon occurs upon PCR amplification, in which the number of repeats is increased or reduced when compared with an original sequence. Such phenomenon is referred to as “stutter”, and provides a negative influence on reliability in the DNA profiling. Therefore, various technologies have been developed in order to eliminate the influence by the stutter. For example, Patent Literature 1 (PTL 1) discloses a technology in which the height of a stutter peak is estimated.
In addition, in a recent DNA profiling, isoalleles which have the same sequence length, but have different nucleotide sequences are identified using a technology referred to as “NGS (next generation sequencing)”. The DNA profiling using NGS reads not only true sequences which have been correctly amplified, but also a stutter sequence generated by stutter. However, the isoalleles are determined by disregarding the stutter sequence in a manner referred to as “stutter filter”. That is, the stutter filter is a filter by which sequences having a read number of a ratio less than a threshold are uniformly disregarded. The read number of the stutter sequence would be significantly smaller than the read number of the true sequences, resulting in disregarding of the stutter sequence.
PTL 1: Tokkai JP 2006-163720A
The following analysis is provided from an aspect of the disclosure. Herein, the disclosure of the PTL is incorporated by reference.
A sample subjected to DNA profiling sometime includes DNAs of multiple persons at different ratios. For example, a sample obtained from a crime scene includes a lot of DNA from a victim and a little of DNA from a criminal offender (hereinafter, referred to as “criminal”). In a case where such sample is analyzed by NGS, the read number of the true sequence from the criminal would be small. If the above described stutter filter is applied thereto, the true sequence from the criminal would be disregarded.
Herein, the technology disclosed in PTL 1 is useful for setting a threshold for the stutter filter, but does not provide any solutions to the above problem.
Accordingly, it is a purpose of the disclosure to provide an information processing apparatus, an information processing method and an information processing program which may contribute to improve the reliability in DNA profiling.
According to a first aspect, there is provided
an information processing apparatus, comprising:
a storage part that stores, for each of isoalleles of a microsatellite which are identified in DNA profiling, a true sequence correctly amplified by PCR, an error sequence incorrectly amplified upon PCR, and a generation probability of the error sequence in association with each other;
an analysis result acquiring part that acquires an analysis result in which read sequences which are read by subjecting a sample to PCR and sequence analysis and read numbers of the read sequences are listed in association with each other;
a prospect part that refers to the storage part while regarding the read sequences as a true sequence for each of the read sequences listed in the analysis result so as to acquire an associated error sequence as a prospected error sequence, and obtains a value as a prospected read number by multiplying the generation probability of the associated error sequence with the read number of each of the read sequences;
a determination part that retrieves a read sequence identical with the prospected error sequence among the read sequences listed in the analysis result, and determines that a retrieved read sequence as an error sequence in a case where the read number of the retrieved read sequence matches with the prospected read number.
According to a second aspect, there is provided an information processing method, including:
an analysis result acquiring step of acquiring an analysis result in which read sequences which are read by subjecting a sample to PCR and sequence analysis and read numbers of the read sequences are listed in association with each other;
a prospect step of referring to a storage part that stores, for each of isoalleles of a microsatellite which are identified in DNA profiling, a true sequence correctly amplified by PCR, an error sequence incorrectly amplified upon PCR, and a generation probability of the error sequence in association with each other, while regarding the read sequences as a true sequence for each of the read sequences listed in the analysis result so as to acquire an associated error sequence as a prospected error sequence, and obtaining a value as a prospected read number by multiplying the generation probability of the associated error sequence with the read number of each of the read sequences;
a determination step of retrieving a read sequence identical with the prospected error sequence among the read sequences listed in the analysis result, and determining that a retrieved read sequence as an error sequence in a case where the read number of the retrieved read sequence matches with the prospected read number.
According to a third aspect, there is provided
an information processing program causing a computer to execute:
an analysis result acquiring process of acquiring an analysis result in which read sequences which are read by subjecting a sample to PCR and sequence analysis and read numbers of the read sequences are listed in association with each other;
a prospect process of referring to a storage part that stores, for each of isoalleles of a microsatellite which are identified in DNA profiling, a true sequence correctly amplified by PCR, an error sequence incorrectly amplified upon PCR, and a generation probability of the error sequence in association with each other, while regarding the read sequences as a true sequence for each of the read sequences listed in the analysis result so as to acquire an associated error sequence as a prospected error sequence, and obtaining a value as a prospected read number by multiplying the generation probability of the associated error sequence with the read number of each of the read sequences;
a determination process of retrieving a read sequence identical with the prospected error sequence among the read sequences listed in the analysis result, and determining that a retrieved read sequence as an error sequence in a case where the read number of the retrieved read sequence matches with the prospected read number.
According to each aspect of the disclosure, there are provided an information processing apparatus, an information processing method and an information processing program that contribute to improve the reliability in DNA profiling.
A preferable example embodiment of the disclosure is explained in detail while referring to drawings. Herein, reference signs appended to the following disclosure expediently appended to each element as one example for an aid for understanding, it is not intended to limit the disclosure to the configuration illustrated in the drawings. In addition, a connection line between blocks in drawings includes both of bidirectional and monodirectional connections. Further, although omitted in block diagrams and the like disclosed in the application, an input port and an output port are provided on an input end and an output end of each connection line, respectively. The same is applied to an input/output interface.
First, terms used in the disclosure are explained. Herein, for example, STRBase (Short Tandem Repeat DNA Internet DataBase, https://strbase.nist.gov/index.htm) should be also referenced for explanation of each term.
“DNA (deoxyribonucleic acid)” refers to a chemical compound comprising adenine (A), guanine (G), cytosine (C) and thymine (T), but also refers to “genetic information” of individual persons in the application. For example, “DNA profiling” may be interchanged by personal profiling based on genetic information, and “DNA of victim” may be interchanged by genetic information of the victim.
“Microsatellite” refers to a repeat sequence itself and a region, a tract, a site, a position which comprise the repeat sequence, but also refers to a comprehensive name of loci in the application.
“Locus (loci)” refers to a position on a chromosome. The locus may be referred to as a marker name, such as CSF1PO, D1S1656 and the like.
“Isoalleles” refers to a type of variants provided on each locus. On the STRBase, it is referred to as Allele (Repeat #): 11′, and the like.
“Sequence” refers to a sequence of nucleotide bases. In addition, “repeat sequence (repetitive sequence)” is also called as STR (short tandem repeat). In a case where a sequence of 2 or more nucleotide bases is regarded as one unit, the “repeat sequence” comprises plural times of repeats of the unit(s) (single or multiple). On the STRBase, it is also referred to as “Repeat Structure”. For example, a repeat sequence indicated by “[CCTA]1[TCTA]10” refers to a sequence in which a unit [TCTA] tandemly repeats 10 times subsequent to a unit [CCTA]. Herein, “[CCTA]1[TCTA]10” may be also indicated as “[TAGA] 10[TAGG]1” (i.e., antiparallel (complementary) sequence), and they are regarded as identical in STR analysis. Herein, there is also a case where 3 to 5 nucleotides are regarded as one repeat unit.
“True sequence” refers to a sequence of a case where a repeat sequence is correctly amplified by PCR (Polymerase Chain Reaction), and “error sequence” refers to a sequence of an incorrectly amplified repeat sequence upon PCR. Herein, “error” includes stutter, indel, nucleotide substitution. That is, the “true sequence” refers to a sequence of which a sequence included in a sample is amplified without any artifacts, such as stutter, etc. Herein, a sequence included in the sample itself may be referred to as both of the “true sequence” and an “original sequence”, but has the same sequence as itself.
“Stutter” refers to a phenomenon that the repeat number is increased or reduced compared with an original sequence upon PCR amplification. Herein, a sequence in which the stutter occurs is referred to as “stutter sequence”.
“Indel (insertion/deletion)” refers to a phenomenon that one or more nucleotide base is inserted into/deleted from an original sequence, and includes indel occurring upon PCR amplification and indel due to artifact upon sequence analysis. Herein, “indel” in the application is used in a different meaning from gene polymorphism within an original sequence (so called insertion/deletion polymorphism). Herein, a sequence in which the indel occurs is referred to as “indel sequence”.
“Nucleotide substitution” refers to a phenomenon that one or more nucleotide base in an original sequence is substituted with another nucleotide base, and includes nucleotide substitution occurring upon PCR amplification and nucleotide substitution due to artifact upon sequence analysis. Herein, “nucleotide substitution” in the application is used as a different meaning from so-called point mutation. Herein, a sequence in which the nucleotide substitution occurs is referred to as “nucleotide substitution sequence”.
“Generation probability of the error sequence” has a similar meaning as those of generation frequency of error, a relative amount of a fragment which is incorrectly amplified upon PCR, and generation frequency of artifact upon sequence analysis.
“Sequential analysis” refers to an analysis for determining a nucleotide sequence, and also refers to as “DNA sequencing”. In addition, “sequential analysis” is also expressed in a context of “reading” a sequence. Herein, the above terms “true sequence”, “error sequence” are also sequences that are determined by the sequential analysis. However, in the application, these sequences have been previously determined by experiments. On the other hand, the term “read sequence” refers to a sequence to be actually read upon DNA profiling, (i.e., raw data).
Herein, in the application, it is preferable that a technology referred to as NGS (next generation sequencing) is applied to the sequential analysis. NGS includes a nanopore sequencing (for example, see WO2016/075204), a cluster generation sequencing (for example, see WO2014/108810), etc. Any types of sequential analysis may be applied to the application, in which DNA fragments are amplified by PCR, sequences of the amplified DNA fragments are read respectively, and then the number of reading of the same sequence (i.e., “read number”) is obtained. In other words, the sequential analysis of the application may be applied if it is possible to finally obtain an analysis result, for example, as shown in
[One Outline of the Disclosure]
Next, one outline of the disclosure is explained while referring to
The storage part 110 stores, for each of isoalleles of a microsatellite which are identified in DNA profiling, a true sequence correctly amplified by PCR, an error sequence incorrectly amplified upon PCR, and a generation probability of the error sequence in association with each other. For example, as illustrated in
The analysis result acquiring part 120 acquires an analysis result in which read sequences which are read by subjecting a sample to PCR and sequence analysis and read number of each of the read sequences are listed in association with each other. For example, the analysis result acquiring part 120 acquires an analysis result illustrated in
The prospect part 130 refers to the storage part 110 while regarding the read sequences as the true sequence for each of the read sequences listed in the analysis result. Then the prospect part 130 acquires an associated error sequence as a prospected error sequence, and obtains a value as a prospected read number by multiplying the generation probability of the associated error sequence with the read number of each of the read sequences.
For example, the prospect part 130 searches the storage part 110, using READ SEQUENCE: [CCTA 1][TCTA 10] as a search key, for a true sequence identical with the read sequence. In the example illustrated in
Furthermore, the prospect part 130 executes the same process for READ SEQUENCE: [TCTA 10], and obtains PROSPECTED ERROR SEQUENCE: [TCTA 9] and PROSPECTED READ NUMBER: 20. With respect to READ SEQUENCES: [CCTA 1][TCTA 9] and [TCTA 9], there are no identical sequences in the true sequences in the storage part 110, thus the prospect part 130 determines PROSPECTED ERROR SEQUENCE: NONE and terminates its process. These processes by the prospect part 130 is conceptionally illustrated in
The determination part 140 retrieves a read sequence identical with the prospected error sequence among the read sequences listed in the analysis result, and determines that the retrieved read sequence as the error sequence in a case where the read number of the retrieved read sequence matches with the prospected read number.
For example, in the example illustrated in
Herein, an effect exerted by the above information processing apparatus 100 is explained while comparing with a case of applying a stutter filter. For example, with respect to LOCUS: D1S1656, it is known that the stutter occurs at a probability of approximately 7%. The stutter filter is a filter for eliminating an effect by the stutter, thus a threshold exceeding 7% (for example 10%) is set as the stutter filter. In a case where 10% is set as the threshold for the stutter filter, in the analysis result of
read sequences having a read number of 1000 or less would be disregarded since the read number of ID: 1 which may be recognized as a true sequence is 10000. That is, in a case where the stutter filter is applied, ID: 2 would be also determined as an error sequence and disregarded. On the other hand, in the information processing apparatus 100 of the disclosure, ID: 2 is determined as a true sequence as indicated in
Such difference provides a significant effect in a case where a sample to be applied to DNA profiling includes DNAs of multiple persons at different rates. For example, a case is considered where a sample which had been obtained from a crime scene and supposed to include a little amount of DNA of a criminal was subjected to PCR and sequential analysis, and then the analysis result illustrated in
If the stutter filter is applied, IDs: 2 to 4 would be determined as the error sequence and disregarded as described above, and only ID: 1 would be determined as the true sequence. ID: 1 would be determined as being derived from a victim, and resulting in a determination that the sample would not include DNA of the criminal.
On the other hand, in the information processing apparatus 100 of the disclosure, ID: 2 is determined as the true sequence. Herein, the read number of ID: 2 is significantly less than the read number of ID: 1, thus it is determined that ID: 2 is derived from a person different from ID: 1. That is, according to the information processing apparatus 100 of the disclosure, ID: 2 is determined as being derived from a criminal.
As described above, according to the information processing apparatus 100 of the disclosure, reliability in DNA profiling may be improved.
In the following description, the information processing apparatus 100 explained in the above one outline is explained more concretely. An information processing apparatus 100 of an example embodiment 1 is realized as a computer comprising a memory, a processor and an interface as illustrated in
Information stored in the storage part 110 may include a plurality of error sequences for one isoallele as illustrated in, for example,
The processor is configured to comprise CPU (Central Processing Unit) and a chip, and reads out programs from the storage part to realize processing modules required for the disclosure. The computer of the example embodiment 1 realizes the analysis result acquiring part 120, the prospect part 130 and the determination part 140 as the processing modules, which are explained in the above one outline. In the following description, points different from the above one outline are explained.
The analysis result acquiring part 120 acquires not only the analysis result relating to LOCUS: D1S1656 as illustrated in
The determination part 140 determines that a read sequence is an error sequence in a case where a read number of a read sequence identical with a prospected error sequence matches with a prospected read number. Herein, the term “match” includes not only a case where the read number of the read sequence is completely consistent with the prospected read number, but also a case where the read number of the read sequence is consistent with the prospected read number at a reasonable extent. For example, in a case where the read number of the read sequence is within ±50% of the prospected read number, the determination part 140 may determine that they match one another. In addition, in a case where the read number of the read sequence is less than the prospected read number, the determination part 140 determines that they match each other. Herein, a range and a threshold in a concept of “match” may be variously set based on, for example, a purpose of DNA profiling, such as paternity test, determination of a criminal, etc., and PCR condition, such as sample condition, PCR condition, etc.
Herein, the determination result provided by the determination part 140 is output and displayed on a display and the like via the interface.
In the following description, a flow of a sequential process by the information processing apparatus 100 of the example embodiment 1 is explained. As illustrated in
As described above, the information processing apparatus 100 of the example embodiment 1 may eliminate, from the DNA profiling, effects due to not only stutter sequence, but also indel sequence and nucleotide substitution sequence generated due to artifact upon PCR.
In an aspect of reliability in DNA profiling, peak height balance in the analysis result would be also regarded as important. An analysis result having imbalanced peak height would provide poor reliability in profiling of a person of heterozygous. Therefore, in the following description, an information processing apparatus 100 capable of overcoming a problem relating to imbalanced peak height is explained as an example embodiment 2. Herein, with respect to the peak height balance, see also for example, Kagaku to Seibutsu 55(8): 559-565 (2017), “Discrimination among Individuals with Analysis of DNA Profiles: Application of New Forensic Science Technologies Using Microbiota Profiling”.
As illustrated in
Herein, the process by the analysis result correcting part 150 have a common concept with a technology referred to as “deblur” in a field of image processing. That is, in the technology referred to as “deblur”, unclear image may be corrected to its original image under a situation where Point spread function is known, which indicates how one point has been spread. Herein, if the “one point” is regarded as the “true sequence”, “how one point has been spread” is regarded as the “error sequence”, and the “Point spread function” is regarded as the generation probability”, the technology referred to as “deblur” may be applied to the process by the analysis result correcting part 150. Herein, with respect to “deblur”, see also Tokuhyo No. 2017-531244, and the like.
An effect by the information processing apparatus 100 of the example embodiment 2 is conceptually explained while referring to a concrete example. For example, a premise is provided, in which a sample was obtained from one person and an analysis result regarding D1S1656 was obtained as illustrated in
In the information processing apparatus 100 of the example embodiment 2, the analysis result correcting part 150 corrects the analysis result illustrated in
That is, it is assumed that the error sequence of ID: 3 is the stutter sequence incorrectly amplified upon PCR amplification of the true sequence of ID: 2, thus, under the assumption that all of the true sequence of ID: 2 would have been correctly amplified, the read number of ID: 2 would be 8000+2000. In addition, assumedly the error sequence of ID: 4 would be the stutter sequence incorrectly amplified upon PCR amplification of the true sequence of ID: 1, thus under the assumption that all of the true sequence of ID: 1 would have been correctly amplified, the read number of ID: 2 would be 10000+400. As described above, the analysis result correcting part 150 corrects the analysis result to indicate the read number of a case where all true sequences are assumed to be correctly amplified.
In the corrected analysis result illustrated in
As described above, according to the information processing apparatus 100 of the example embodiment 2, the read number of the error sequence incorrectly amplified upon PCR amplification is added to the read number of the true sequence, thus peak height balance is improved. As a result, according to the information processing apparatus 100 of the example embodiment 2, reliability in DNA profiling is improved for a profile regarding a person having heterozygote.
A part or all of the example embodiments are described as the following modes, but not limited thereto.
(Mode 1)
An information processing apparatus, comprising:
a storage part that stores, for each of isoalleles of a microsatellite which are identified in DNA profiling, a true sequence correctly amplified by PCR, an error sequence incorrectly amplified upon PCR, and a generation probability of the error sequence in association with each other;
an analysis result acquiring part that acquires an analysis result in which read sequences which are read by subjecting a sample to PCR and sequence analysis and read numbers of the read sequences are listed in association with each other;
a prospect part that refers to the storage part while regarding the read sequences as a true sequence for each of the read sequences listed in the analysis result so as to acquire an associated error sequence as a prospected error sequence, and obtains a value as a prospected read number by multiplying the generation probability of the associated error sequence with the read number of each of the read sequences;
a determination part that retrieves a read sequence identical with the prospected error sequence among the read sequences listed in the analysis result, and determines that a retrieved read sequence as an error sequence in a case where the read number of the retrieved read sequence matches with the prospected read number.
(Mode 2)
The information processing apparatus according to Mode 1, wherein the determination part determines a read sequence which is not determined as the error sequence among the read sequences listed in the analysis result as a true sequence.
(Mode 3)
The information processing apparatus according to Mode 1 or 2, further comprising an analysis result correcting part that corrects the analysis result in a manner that the read number of the read sequence determined as the error sequence by the determination part is added to the read number of the read sequence regarded as a true sequence.
(Mode 4)
The information processing apparatus according to any one of Modes 1 to 3, wherein the error sequence is: a stutter sequence in which repeat number is increased or reduced when compared with an original sequence; an indel sequence in which one or more nucleotide base is inserted into/deleted from an original sequence; and/or a nucleotide substitution sequence in which at least one nucleotide base in an original sequence is substituted with another nucleotide base.
(Mode 5)
An information processing method, including:
an analysis result acquiring step of acquiring an analysis result in which read sequences which are read by subjecting a sample to PCR and sequence analysis and read numbers of the read sequences are listed in association with each other;
a prospect step of referring to a storage part that stores, for each of isoalleles of a microsatellite which are identified in DNA profiling, a true sequence correctly amplified by PCR, an error sequence incorrectly amplified upon PCR, and a generation probability of the error sequence in association with each other, while regarding the read sequences as a true sequence for each of the read sequences listed in the analysis result so as to acquire an associated error sequence as a prospected error sequence, and obtaining a value as a prospected read number by multiplying the generation probability of the associated error sequence with the read number of each of the read sequences;
a determination step of retrieving a read sequence identical with the prospected error sequence among the read sequences listed in the analysis result, and determining that a retrieved read sequence as an error sequence in a case where the read number of the retrieved read sequence matches with the prospected read number.
(Mode 6)
An information processing program causing a computer to execute:
an analysis result acquiring process of acquiring an analysis result in which read sequences which are read by subjecting a sample to PCR and sequence analysis and read numbers of the read sequences are listed in association with each other;
a prospect process of referring to a storage part that stores, for each of isoalleles of a microsatellite which are identified in DNA profiling, a true sequence correctly amplified by PCR, an error sequence incorrectly amplified upon PCR, and a generation probability of the error sequence in association with each other, while regarding the read sequences as a true sequence for each of the read sequences listed in the analysis result so as to acquire an associated error sequence as a prospected error sequence, and obtaining a value as a prospected read number by multiplying the generation probability of the associated error sequence with the read number of each of the read sequences;
a determination process of retrieving a read sequence identical with the prospected error sequence among the read sequences listed in the analysis result, and determining that a retrieved read sequence as an error sequence in a case where the read number of the retrieved read sequence matches with the prospected read number.
Herein, it is considered that the disclosures of the above Patent Literatures and cited literatures are incorporated herein by reference thereto, and the disclosures may be used as a base or a part of the disclosure as necessary. Variations and adjustments of the example embodiments and examples are possible within the ambit of the entire disclosure (including the claims) of the disclosure and based on the basic technical concept thereof. In addition, various combinations and selections (including non-selection) of various disclosed elements (including each element in each claim, each example embodiment, each drawing, etc.) are possible within the ambit of claims of the disclosure. Namely, the disclosure of course includes various variations and modifications that could be made by those skilled in the art according to the overall disclosure including the claims and the technical concept. Further, each of the disclosed matters of the above cited literatures is regarded as included in the described matters in the application, if required, on the basis of the concept of the disclosure, as a part of the disclosure, also that a part or entire thereof is used in combination with a described matter(s) in the application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-102716 | May 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/021351 | 5/29/2020 | WO | 00 |
