JPWO2016114009A1 - Fusion gene analysis apparatus, fusion gene analysis method, and program - Google Patents

Abstract

A lead sequence acquisition unit that acquires the read sequence output from the sequencer, a complementary sequence generation unit that generates complementary sequences for all the acquired read sequences and outputs them as virtual complementary sequences, and mapping the read sequences and virtual complementary sequences A mapping information acquisition unit that supplies the device and acquires a mapping result on the reference sequence by the mapping device; in the mapping result, the lead sequence and the corresponding virtual complementary sequence are each divided into two portions of the reference sequence and mapped A candidate lead sequence extraction unit that extracts a candidate read sequence as a candidate lead sequence, and uses a division point of the extracted candidate lead sequence as a breakpoint candidate; Group creation unit for grouping arrays into one group, and candidates for configuring the group A fusion gene determination information generation unit that generates information for determining whether or not the candidate lead sequences included in each group are derived from a fusion gene based on the characteristics and number of card sequences; Prepare.

Description

  The present invention relates to a fusion gene analyzer, a fusion gene analysis method, and a program.

  In recent years, fusion genes have attracted attention in cancer treatment. For example, in Patent Document 1, genome data of a patient obtained from a sequencing machine is analyzed using a data source distributed on a network, and the position of a mutation related to cancer and the result of the mutation are disclosed. As a system for providing information on the disease that occurs as a result.

JP 2014-146318 A

  However, the method described in Patent Document 1 has a problem in that it takes a long time to analyze the genome data obtained from the sequencing machine without any particular restriction. Also, since there is no processing to eliminate analysis errors at the sequencing or alignment stage, the accuracy of analysis is not sufficient. Further, Patent Document 1 does not fully describe detection and extraction of a fusion gene.

  Therefore, an object of the present invention is to realize improvement in accuracy and time reduction in fusion gene analysis.

The fusion gene analysis system according to the present invention includes a read sequence acquisition unit that acquires a read sequence output from a sequencer, and a virtual complementary sequence generation unit that generates complementary sequences for all the acquired read sequences and outputs them as virtual complementary sequences A mapping information acquisition unit that supplies the lead sequence and the virtual complementary sequence to a mapping device and acquires a mapping result on a reference sequence by the mapping device; and in the mapping result, the lead sequence and the corresponding virtual complement A candidate lead sequence extraction unit that extracts a sequence in which the sequence is divided and mapped in two portions of the reference sequence as a candidate lead sequence, and uses a division point of the extracted candidate lead sequence as a breakpoint candidate; , A candidate read sequence in which the breakpoint candidate is in the vicinity within a predetermined number of bases In order to determine whether or not the candidate lead sequences included in each group are derived from a fusion gene, based on the group creation unit to be grouped into one group and the characteristics and number of candidate lead sequences constituting the group And a fusion gene judgment information generation unit for generating the information.

  The candidate lead sequence extraction unit may extract a candidate lead sequence in which the corresponding fragment of the lead sequence mapped to two locations and the virtual complementary sequence are mapped on the same chromosome. Good.

In addition, the candidate lead sequence extraction unit,
A candidate lead sequence may be extracted in which the fragments of the lead sequence and the virtual complementary sequence mapped at two locations have a length equal to or longer than a predetermined number of bases.

  The fusion gene determination information generation unit may rank each group according to the possibility that the candidate lead sequence included in each group is derived from the fusion gene.

Further, the fusion gene determination information generation unit may set a higher rank for a group having a large number of candidate lead sequences constituting the group.
In addition, the fusion gene determination information generation unit sets the rank of the group low when there is a predetermined number or more of the other division points with respect to one division point of the candidate lead sequence constituting the group. You may do it.

  The fusion gene analyzing method according to the present invention includes a step of obtaining a lead sequence output from a sequencer, a step of creating a complementary sequence for all the obtained lead sequences, and outputting as a virtual complementary sequence, the lead sequence and the Supplying a virtual complementary sequence to a mapping device and obtaining a mapping result onto a reference sequence by the mapping device; and in the mapping result, the lead sequence and the corresponding virtual complementary sequence are each two of the reference sequences. A segmented and mapped segment is extracted as a candidate lead sequence, and a segmentation point of the extracted candidate lead sequence is set as a breakpoint candidate, and the breakpoint candidate is in the vicinity of a predetermined number of bases A step of grouping candidate lead sequences into one group, and a candidate library constituting the group Based on the characteristics and number of sequences, it is intended to include the steps of candidate lead sequence included in each of the groups to generate information for determining whether or not derived from a fusion gene, a.

A program according to the present invention includes a read sequence acquisition unit that acquires a read sequence output from a sequencer, and a virtual complementary sequence generation unit that generates complementary sequences for all the acquired read sequences and outputs them as virtual complementary sequences A mapping information acquisition unit that supplies the lead sequence and the virtual complementary sequence to a mapping device and acquires a mapping result on a reference sequence by the mapping device; and in the mapping result, the lead sequence and the corresponding virtual complement A candidate lead sequence extraction unit that extracts a sequence in which the sequence is divided and mapped in two portions of the reference sequence as a candidate lead sequence, and uses a division point of the extracted candidate lead sequence as a breakpoint candidate; Candidate reads where the breakpoint candidate is in the vicinity of a predetermined number of bases Based on the characteristics and number of candidate read sequences constituting the group, and a group creation unit that groups the columns into one group, it is determined whether the candidate read sequences included in each group are derived from a fusion gene. It is made to function as a fusion gene judgment information generation part which produces | generates the information for doing.

According to the present invention, it is possible to improve the accuracy of fusion gene analysis and shorten the time.

The figure which shows the outline | summary of the fusion gene analysis system by embodiment of this invention. The block diagram which shows the structure of the fusion gene analyzer by embodiment of this invention. The figure explaining the mapping result by embodiment of this invention. The flowchart of operation | movement of the fusion gene analysis system by embodiment of this invention. The figure which shows the result of the analysis by the fusion gene analysis system by embodiment of this invention.

Next, embodiments for carrying out the present invention will be described in detail with reference to the drawings.
FIG. 1 is a block diagram showing a configuration of a fusion gene analysis system 10 according to an embodiment of the present invention. As shown in the figure, the fusion gene analysis system 10 includes a fusion gene analysis device 100, a DNA sequencer 200, and a gene mapping device 300. The fusion gene analyzing apparatus 100, the DNA sequencer 200, and the gene mapping apparatus 300 are connected via a communication line 50.

  FIG. 2 is a block diagram showing the configuration of the fusion gene analyzing apparatus 100. As shown in the figure, the fusion gene analyzing apparatus 100 includes a lead sequence acquisition unit 101, a virtual complementary sequence generation unit 102, a mapping information acquisition unit 103, a candidate lead sequence extraction unit 104, a group creation unit 105, and a fusion gene determination information generation unit. 106, a display device 107, and an input device 108.

  The fusion gene analyzing apparatus 100 applies a dedicated or general-purpose computer including a CPU, a memory such as a ROM or a RAM, an external storage device for storing various information, an input interface, an output interface, a communication interface, and a bus connecting them. Can do. The fusion gene analyzing apparatus 100 may be configured by a single computer or may be configured by a plurality of computers connected to each other via a communication line.

  The lead sequence acquisition unit 101, the virtual complementary sequence generation unit 102, the mapping information acquisition unit 103, the candidate lead sequence extraction unit 104, the group creation unit 105, and the fusion gene determination information generation unit 106 have a predetermined CPU stored in a ROM or the like. This corresponds to a function module realized by executing a program.

The display device 107 is a display device such as a display, and displays various images in response to an image signal output from the CPU of the fusion gene analyzer 100.
The input device 108 is various devices including a mouse, a keyboard, and the like, and is used when a user inputs various information to the fusion gene analysis device 100.

  The read sequence acquisition unit 101 acquires the read sequence output from the DNA sequencer 200. The DNA sequencer 200 may be of a single end read type or a pair end read type. Further, genomic DNA is used as a sample used for base sequence analysis. Furthermore, analysis efficiency can be increased by using a target capture sample obtained by amplifying only the base sequence of a specific region.

  The virtual complementary sequence generation unit 102 creates a complementary sequence for all the acquired read sequences and outputs it as a virtual complementary sequence. Specifically, the virtual complementary sequence generation unit 102 takes as input a read sequence composed of sequences of bases A (adenine), T (thymine), G (guanine), and C (cytosine), and complements each base. The basic bases (A → T, T → A, G → C, C → G) and rearranged in reverse order are output as virtual complementary sequences.

  As a result, even when the DNA sequencer 200 of the single end read method is used, mapping can be performed using a set of the read sequence and the complementary sequence as in the pair end read method. Furthermore, in this embodiment, since a virtual complementary sequence is created for all the acquired read sequences, not only a limited range of complementary sequences can be obtained as in the general paired-end read method, but also the read sequence. The entire complementary sequence can be obtained.

  The mapping information acquisition unit 103 supplies the lead sequence and the virtual complementary sequence to the gene mapping apparatus 300, and acquires the result of mapping onto the reference sequence by the gene mapping apparatus 300. The mapping apparatus 300 maps the lead sequence and the virtual complementary sequence onto the reference sequence by, for example, a BWA (Burrows-Wheeler Alignment) -SW (smith-waterman) algorithm.

  The candidate lead sequence extraction unit 104 extracts, as a candidate lead sequence, a mapping result obtained by dividing the mapping of the lead sequence and the corresponding virtual complementary sequence into two portions of the reference sequence. The division point of the lead array is set as a breakpoint candidate.

  Fusion genes include chromosomal translocations in which the position of the base sequence is changed between chromosomes, interstitial deletions in which part of the base sequence in the chromosome disappears, and chromosomal inversions in which the position of the base sequence is changed in the same chromosome. It is formed by the fusion of genes that were originally separated.

  If the lead sequence contains a fusion gene, the mapping result shows that the lead sequence derived from the fusion gene is divided into two, with the fusion point (breakpoint) as the boundary, and each fragment is located at a different position on the reference sequence. To be mapped. The virtual complementary sequence is also divided with the same breakpoint as a boundary, and each fragment is mapped to the same position on the reference sequence as the corresponding lead sequence. FIG. 3 is a diagram illustrating an example of the mapping result.

  As shown in FIG. 3, when the lead sequence (r1) and the virtual complementary sequence (r1 ′) are each divided into two locations, and the corresponding sequences are mapped to the same region, the candidate lead sequence extraction unit 104 Extracts the lead sequence as a candidate lead sequence. Further, two division points (b1, b2) of the candidate read sequence are set as breakpoint candidates.

  Note that when the candidate lead sequence extraction unit 104 extracts candidate lead sequences, only those satisfying the following conditions (A) to (D) may be extracted.

(A) The candidate lead sequence extraction unit 104 maps the divided lead sequence and the virtual complementary sequence only in two places (a total of four places in the lead sequence and the virtual complementary sequence), and fragments in two places Only when a complete lead sequence or a virtual complementary sequence is obtained by combining the two, a candidate lead sequence may be used.

(B) The candidate lead sequence extraction unit 104 extracts, as candidate lead sequences, those in which corresponding fragments of the lead sequence mapped to two locations and the virtual complementary sequence are mapped on the same chromosome. Also good.

(C) The candidate lead sequence extracting unit 104 reads each candidate sequence in which each of the lead sequence mapped to two locations and the virtual complementary sequence has a length of a predetermined number of bases or more (for example, 10 bases or more). You may make it extract as an arrangement | sequence. Thereby, what is divided | segmented by the variation | mutation etc. of only 1 base can be excluded.

(D) The candidate lead sequence extraction unit 104, when the fusion gene is a chromosomal inversion in which the position of the base sequence is interchanged within the same chromosome, determines that the two breakpoint candidates are separated by 1 million bases or more. May be extracted as

The group creation unit 105 collects candidate read sequences having breakpoint candidates in the vicinity within a predetermined number of bases into one group.
In FIG. 3, the candidate lead sequences r2 to r4 have breakpoint candidates at substantially the same positions as the candidate lead sequence r1. In such a case, the group creation unit 105 groups the candidate lead arrays r1 to r4 into one group as having the same breakpoint candidates. Specifically, for example, if the dividing points of the candidate read sequences are the same within an error range of 40 bases, the same group may be used.

  The fusion gene determination information generation unit 106 is information for determining whether or not the candidate lead sequences included in each group are derived from the fusion gene based on the characteristics and number of candidate lead sequences constituting the group. (Ranking) is generated.

  First, the fusion gene determination information generation unit 106 ranks each group according to the possibility that the candidate lead sequence included in each group is derived from the fusion gene. Specifically, for each group, it is determined whether or not the following four narrowing conditions are satisfied.

(1) On gene determination The fusion gene determination information generation unit 106 determines whether or not there is an overlap with a gene region for two fragments obtained by dividing the lead sequence of each group. If both fragments overlap with the gene region, it is determined that the condition is satisfied (high possibility of being a fusion gene).

(2) Known Target Gene Determination The fusion gene determination information generation unit 106 is a gene in which, for a group satisfying the condition (1), two fragments obtained by dividing the lead sequence are known as genes constituting the fusion gene. It is determined whether it corresponds to. Specifically, if a receptor tyrosine kinase gene such as RET, ROS1, or ALK is included, it is determined that the condition is satisfied. These kinase genes are effective in determining fusion genes and are effective in selecting therapeutic agents.

(3) In frame determination The fusion gene determination information generation unit 106 determines whether or not a frame shift has occurred in the exon region of the fragment of the lead sequence for the group satisfying the condition (2). If no frame shift has occurred, it is determined that the condition is satisfied. When a frameshift occurs in the exon region, it is considered that the protein is not synthesized, so that it is not very suitable as a target for cancer treatment.

(4) Coiled-Coil structure determination The fusion gene determination information generation unit 106 determines whether the gene upstream of the lead sequence fragment has a Coiled-Coil structure for the group satisfying the condition (3), When it has a Coiled-Coil structure, it determines with satisfy | filling conditions. For example, many gene fragments fused to receptor tyrosine kinase genes such as RET, ROS1, and ALK have a Coiled-Coil structure that causes protein-protein interactions, and they serve as ligands that transmit growth signals from outside the cell. It is known to activate kinases independently.

  The fused gene determination information generation unit 106 assigns higher ranks to groups satisfying many determination conditions for the determination conditions (1) to (4). It should be noted that ranking may be performed using only some of the conditions (for example, (1) and (2)) without performing determination for all the above conditions.

  Next, the fused gene determination information generation unit 106 sets the rank of the group having a large number of candidate lead sequences constituting the group to be high. For example, the higher the number of candidate read sequences, the higher the rank. Also, if there is more than a certain number of other division points for one division point of the candidate lead sequence constituting the group, the non-specificity of mapping is suspected, so the rank should be set low. Also good. For example, within a group, there are a candidate lead sequence having a set of dividing points (b1, b2) shown in FIG. 3 and a candidate lead sequence having a set of dividing points (b3, b4), and b1 and b3 are close to each other. However, when b2 and b4 are separated, the rank of the group may be set low.

  The fusion gene determination information generation unit 106 ranks all groups based on ranking based on the narrowing-down conditions and ranking based on the number of candidate lead sequences constituting the group. For example, the ranking based on the narrowing-down condition and the ranking based on the number of candidate lead sequences may be used as points, and the higher the overall points, the higher the rank may be. The fusion gene determination information generation unit 106 displays a list in which the candidate lead sequences are arranged in descending order on the display device 107.

Next, the operation of the fusion gene analysis system 10 will be described.
FIG. 4 is a flowchart of the operation of the fusion gene analysis system 10.
First, the read sequence acquisition unit 101 acquires a read sequence from the DNA sequencer 200 (step S1).
Next, the virtual complementary sequence generation unit 102 creates a complementary sequence for all the acquired read sequences and outputs it as a virtual complementary sequence (step S2).

Next, the mapping information acquisition unit 103 inputs the lead sequence and the virtual complementary sequence to the gene mapping apparatus 300 (step S3).
Next, the gene mapping apparatus 300 performs mapping between the input read sequence and the virtual complementary sequence (step S4).
Next, the mapping information acquisition part 103 acquires the result of the mapping by the gene mapping apparatus 300 (step S5).

Next, the candidate lead sequence extraction unit 104 extracts a candidate lead sequence from the mapping result by the gene mapping apparatus 300 (step S6).
Further, the candidate lead sequence extraction unit 104 sets breakpoint candidates for the extracted candidate lead sequence (step S7).

Next, the group creation unit 105 collects candidate read sequences having breakpoint candidates in the vicinity within a predetermined number of bases into one group (step S8).
Next, the fusion gene determination information generation unit 106 ranks the possibility that the candidate lead sequence included in each group is derived from the fusion gene using the fusion gene narrowing conditions (step S9).

Next, the fusion gene determination information generation unit 106 ranks each group based on the number of candidate lead sequences constituting the group (step S10).
Furthermore, the fusion gene determination information generation unit 106 ranks all groups based on the ranking based on the narrowing-down condition and the ranking based on the number of candidate lead sequences constituting the group, and displays the result on the display device 107. (Step S11).

  As described above, according to the present embodiment, virtual complementary sequences are created for all the read sequences output from the sequencer, and candidate read sequences are extracted based on the mapping result between the read sequence and the virtual complementary sequence. Therefore, since the mapping is performed with the double of the lead sequence and the virtual complementary sequence, the accuracy of mapping can be improved and the accuracy of fusion gene analysis can also be improved.

  In addition, candidate lead sequences that are close to breakpoint candidates are grouped into one group, and the candidate lead sequences are further narrowed down based on the characteristics and number of candidate lead sequences constituting the group. It is possible to limit the number of lead sequences with high accuracy, thereby improving the efficiency of fusion gene analysis and shortening the time.

(Example)
FIG. 5 is a diagram showing the results of analysis by the fusion gene analysis system 10.
In the example of FIG. 5, MiSeq (manufactured by Illumina) and Ion Torrent (manufactured by Thermo Fisher Scientific) are used as the DNA sequencer 200, and three types of cell lines AD09-232T (positive for ALK-EML4 fusion gene) The results of analyzing HCC78 (ROS1-SCL34A2 fusion gene positive) and LC2 / ad (CCDC6-RET fusion gene positive) samples are shown. MiSeq is a pair-end read sequencer, and Ion Torrent is a single-end read sequencer.

  The “total number of reads” of the input data indicates the number of read sequences output from the DNA sequencer 200. The “lead sequence / virtual complementary sequence” is the total number of the lead sequence and the virtual complementary sequence created by the virtual complementary sequence generation unit 102, and corresponds to twice the total number of reads. The “mapping result” indicates the total number of reads after mapping by the gene mapping apparatus 300 (the total of the read sequence and the virtual complementary sequence). Here, mapping is performed by the BWA-SW method.

  The “classification according to the number of map locations” shows the result of classifying each lead according to whether the virtual complementary sequence corresponding to the lead sequence is mapped together. As described above, two virtual complementary sequences corresponding to the lead sequence are mapped to two locations, that is, a total of “four locations”, and are extracted as candidate lead sequences. Furthermore, the number of candidate lead sequences finally extracted by the candidate lead sequence extraction unit 104 is narrowed down under the above-described predetermined conditions, and is indicated as “number of candidate lead sequences”.

  Furthermore, the number of groups collected by the group creation unit 105 is indicated as “number of groups”. Furthermore, the number of candidate lead sequences determined by the fusion gene determination information generation unit 106 to satisfy the On gene determination condition is indicated as “On gene candidate number”. Furthermore, among the On gene candidates, the number of candidate read sequences determined by the fusion gene determination information generation unit 106 to satisfy the known target gene determination condition is indicated as “RET / ROS1 / ALK candidate number”. . Looking at the “number of RET / ROS1 / ALK candidates”, the lead sequences that are candidates for fusion genes are narrowed down to 639,924,271 for each sample.

  As described above, for each sample, the number of candidate fusion gene reads can be greatly reduced from the total number of reads output from the sequencer.

  DESCRIPTION OF SYMBOLS 10 Fusion gene analysis system, 50 communication line, 100 Fusion gene analyzer, 101 Lead sequence acquisition part, 102 Virtual complementary sequence generation part, 103 Mapping information acquisition part, 104 Candidate lead sequence extraction part, 105 Group preparation part, 106 Fusion gene Judgment information generation unit, 107 display device, 108 input device, 200 DNA sequencer, 300 gene mapping device

Claims (8)

A read array acquisition unit for acquiring a read array output from the sequencer;
A virtual complementary sequence generation unit that creates a complementary sequence for all of the obtained read sequences and outputs as a virtual complementary sequence;
A mapping information acquisition unit that supplies the lead sequence and the virtual complementary sequence to a mapping device, and acquires a mapping result on a reference sequence by the mapping device;
In the mapping result, the lead sequence and the corresponding virtual complementary sequence are divided and mapped to two locations of the reference sequence, respectively, and extracted as candidate lead sequences, and the division points of the extracted candidate lead sequences are extracted A candidate lead sequence extraction unit,
A group creation unit that groups candidate read sequences in which the breakpoint candidates are in the vicinity of a predetermined number of bases into one group;
Fusion gene judgment information for generating information for judging whether or not the candidate lead sequences included in each group are derived from a fusion gene based on the characteristics and number of candidate lead sequences constituting the group A fusion gene analyzer comprising: a generation unit; The candidate lead sequence extraction unit includes:
The fusion gene analyzing apparatus according to claim 1, wherein the lead sequence mapped to two locations and the corresponding fragment of the virtual complementary sequence are extracted as candidate lead sequences, which are mapped on the same chromosome. The candidate lead sequence extraction unit includes:
The fusion gene analyzing apparatus according to claim 1, wherein each of the lead sequence and the virtual complementary sequence fragment mapped in two locations is extracted as a candidate lead sequence having a length equal to or longer than a predetermined number of bases. The fusion gene judgment information generation unit
The fusion gene analysis apparatus according to claim 1, wherein each group is ranked according to the possibility that the candidate lead sequence included in each group is derived from the fusion gene. The fusion gene judgment information generation unit
The fusion gene analyzing apparatus according to claim 4, wherein a rank of a group having a large number of candidate read sequences constituting the group is set high. The fusion gene judgment information generation unit
The fusion gene analyzing apparatus according to claim 4, wherein when there is a predetermined number or more of the other dividing points with respect to one dividing point of the candidate lead sequence constituting the group, the rank of the group is set low. . Obtaining a read sequence output from the sequencer;
Creating a complementary sequence for all the obtained read sequences and outputting it as a virtual complementary sequence;
Supplying the lead sequence and the virtual complementary sequence to a mapping device, and obtaining a mapping result on a reference sequence by the mapping device;
In the mapping result, the lead sequence and the corresponding virtual complementary sequence are divided and mapped to two locations of the reference sequence, respectively, and extracted as candidate lead sequences, and the division points of the extracted candidate lead sequences are extracted A process for making a breakpoint candidate,
Grouping candidate read sequences in which the breakpoint candidates are in the vicinity within a predetermined number of bases into one group;
Generating information for determining whether or not the candidate lead sequences included in each group are derived from a fusion gene based on the characteristics and number of candidate lead sequences constituting the group. Fusion gene analysis method. Computer
A read array acquisition unit for acquiring a read array output from the sequencer;
A virtual complementary sequence generation unit that creates a complementary sequence for all of the obtained read sequences and outputs as a virtual complementary sequence;
A mapping information acquisition unit that supplies the lead sequence and the virtual complementary sequence to a mapping device, and acquires a mapping result on a reference sequence by the mapping device;
In the mapping result, the lead sequence and the corresponding virtual complementary sequence are divided and mapped to two locations of the reference sequence, respectively, and extracted as candidate lead sequences, and the division points of the extracted candidate lead sequences are extracted A candidate lead sequence extraction unit,
A group creation unit that groups candidate read sequences in which the breakpoint candidates are in the vicinity of a predetermined number of bases into one group;
Fusion gene judgment information for generating information for judging whether or not the candidate lead sequences included in each group are derived from a fusion gene based on the characteristics and number of candidate lead sequences constituting the group A program that functions as a generator.

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