JP4557609B2 - How to display splice variant sequence mapping - Google Patents

Description

  The present invention relates to information analysis of gene sequences, and more particularly, to analysis of correlation between exon / intron structure of a large amount of splice variant sequences, expression information, and transcription start position.

  In eukaryotes including humans, various mRNAs having different sequences are generated by the same primary transcript from one gene region on the genome through different splicing processes. They are called splice variants. They produce proteins with different sequences and thus perform different functions in vivo. In addition, there are a promoter region and a regulatory region upstream of the gene region on the genome sequence, and these regions control when and under what conditions the gene starts transcription. . One gene may generate a protein having a different sequence by changing its transcription start position, and may perform different functions in vivo. These are one important in vivo mechanism for giving diversity to the genes due to differences in the tissues to be expressed or developmental stages.

  Sequence data of mRNA expressed in vivo can be obtained by preparing a cDNA library and reading the base sequence with a sequencer. Since there is a limit to the number of bases that can be read at one time by a sequencer, it is usually costly to repeatedly sequence in order to obtain a sequence over the entire length of mRNA. Therefore, in order to easily obtain information on what genes are expressed due to differences in the tissues to be expressed or developmental stages, only a part of the mRNA sequence was read once with a sequencer. There are many known EST (Expressed Sequence Tags) sequences or single-pass sequences.

  MRNA sequences that are different from each other as splice variants have different exon-intron structures, and in some cases, may have different transcription start positions. The exon / intron structure and transcription start position of the mRNA sequence are determined by mapping the sequence onto the genome sequence using a sequence similarity search program (Non-patent Document 4). The exon sequence is identified as a homologous section between the genomic sequence and the mRNA sequence, and the intron part is identified as a section in the genomic sequence sandwiched between the exon sequences. The transcription start position is identified as the position where the 5 'end of the mRNA sequence is mapped on the genome.

  The result of mapping the mRNA sequence to the genome sequence is usually displayed by arranging exon sequences along the genome sequence (Non-Patent Documents 1, 2, and 3). This display helps to understand the difference as a splice variant. In general, intron sequences are very long compared to exon sequences, so in order to visualize differences as splice variants more effectively, compress the common intron sequence, and then align the exon sequence along the genome sequence. There is also a display method of arranging them (Patent Document 1).

JP 2003-256434 A The NCBI Handbook, 2002 Nov. Part3, chapter 20, p20-16http: //www.ncbi.nlm.nih.gov/entrez/query.fcgi? Db = Books A User's Guide to the Human Genome.Wolfsberg, T.G., Wetterstrand, K.A., Guyer, M.S., Collins, F.S., and Baxevanis, A.D. (2004). Yutaka Suzuki, Riu Yamashita, Sumio Sugano, and Kenta Nakai, DBTSS, DataBase of Transcriptional Start Sites: progress report 2004, Nucl. Acids. Res. 2004 32: D78-D81 Florea, L. et al., A Computer Program for Aligning a cDNA Sequence with a Genomic DNA Sequence, Genome Research 8: 967-974, 1998.

  In order to investigate how gene expression changes due to differences in many tissues in the body, the presence or absence of disease, the stage of development, the elapsed time when external stimuli are applied, etc. A large amount of EST sequence data of 10,000 or more is known. When these are mapped on the genome, thousands to tens of thousands of EST sequences may be mapped to specific locations on the genome. In such places, the conventional mapping result visualization method has to draw a large number of line segments in proportion to the number of EST sequences, which adds to the display cost and increases the amount of data displayed by humans. At first glance, it becomes difficult to understand what types of splice variants exist. In addition, for biological interpretation, it is important to examine the splicing or transcription start position depending on the gene expression conditions, but in the conventional visualization method, classification as a splice variant or Since the relationship between the transcription start position and the expression information is not clearly shown, it has been difficult to find such a relationship.

  An object of the present invention is to provide a splice variant display method capable of easily displaying the type of splice variant and the relationship between transcription start position and expression information.

  The present invention visualizes the type difference as a splice variant for a large amount of EST sequences and full-length mRNA sequences, visualizes expression information for each type, and visualizes expression information for each transcription start position. It consists of the following processing steps. Hereinafter, the EST sequence or the full-length mRNA sequence is collectively referred to as a cDNA sequence.

  mapping by a sequence similarity search between the cDNA sequence and the genome sequence, and determining the exon / intron structure and transcription start position of each cDNA sequence.

  comparing exon-intron structures of cDNA sequences and classifying the cDNA sequences into types presumed to be derived from the same splice variant.

  The step of collecting cDNA sequences belonging to the same type and visualizing the exon / intron structure along the genome sequence for each type.

  Collecting expression information of cDNA sequences classified into the same type and displaying a breakdown of the expression information for each type;

  Obtaining a transcription start position of a cDNA sequence containing a 5 'end, and collecting cDNA sequences having the transcription start position at each position on the genome sequence;

  A step of collecting expression information of a cDNA sequence having a transcription start position at each position on the genome sequence and displaying a breakdown of the expression information for each transcription start position.

  Since the amount of plotting when visualizing the mapping result is proportional to the number of types that classify the cDNA sequences, not the number of cDNA sequences, for example, drawing for millions of cDNA sequence data using the conventional display method The amount is suppressed to a few tenths. As a result, analysis results for a large amount of sequence data can be displayed in a compact manner, and the amount of data that should be viewed by humans is reduced, making it easier to understand. In addition, since the difference in expression information for each type and the difference in expression information for each transcription start position are explicitly indicated, splicing or transcription initiation is performed according to biological interpretation, that is, the difference in gene expression conditions. It becomes easy to check that the position changes.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 shows an example of displaying the result of splice variant analysis according to the present invention for a large amount of cDNA sequences.

  In FIG. 1, 101 is a visualization of each type of exon / intron structure when a cDNA sequence derived from one gene region is classified as a splice variant according to a known method (Patent Document 1). 102 is the genome sequence coordinate compressed by removing the intron sequence common to the splice variant from within the genome sequence. 103 is a coordinate for arranging each type classified as a splice variant. A line segment 104 represents one exon included in one type. One type of exon-intron structure is represented by arranging several 104 line segments representing exons along the coordinates 102. At that time, end points are clearly shown at both ends of the line segment so that the connected line segments can be distinguished from each other. A gap between line segments 104, such as 105, is another type of splice variant where another exon is inserted there, or one or both of the exons represented by the left and right line segments 104 This indicates that the base sequence can be inserted by changing the splice site. Such representations for all types are aligned along the coordinates 103. The cDNA sequences having no splice sites are collectively shown as 106 with a range that can be taken along the coordinate 102.

  Reference numeral 107 represents expression information for each type along the coordinates of 103. 108 is a coordinate indicating the expression intensity. 109 represents the expression information for one type. The breakdown of the expression intensity for each classification of the expression information is indicated by shading or color, and at the same time, the expression intensity of that type is indicated by the total. Similarly, 110 represents expression information for the entire cDNA sequence without a splice site.

  111 represents the expression information for each transcription start position along the coordinates of 102. 112 is a coordinate representing the expression intensity. 113 represents expression information for one transcription start position. The breakdown of the expression intensity for each classification of the expression information is indicated by shading or color, and at the same time, the expression intensity of the transcription start position is indicated by the total.

  Reference numeral 114 denotes a list box for selecting one gene region displayed in 101. In each element of the list, a gene region name (cluster name) indicated by 115 and gene region annotation information indicated by 116 are displayed. Examples of gene region annotation include detailed positional information on the chromosome of the gene region, and a collection of annotations for full-length mRNA sequences belonging to the gene region (cluster). 117 is a marker indicating the element of the currently selected list box. In the list box 114, clusters that have been narrowed down, rearranged, or selected according to the conditions described in the search window 118 are displayed. The search condition includes a chromosome number, a position on the chromosome, a name of a gene to be included in the cluster, a part of the annotation information, or a condition regarding expression information of the cluster or the transcription start position. The selection of 117 is determined by looking at the contents of the list box 114 displayed as a result of the user's input to the search window 118 and reselecting the items in the list box as necessary. The gene region selected in 117 is displayed in 101.

  FIG. 5 is a schematic configuration diagram of a splice variant analysis / display device according to the invention. This splice variant analysis / display device includes an operation / input unit 501 that performs operations and inputs to the processing unit 503, a display unit 502 that displays processing results, and a storage unit 504 that stores data and information to be processed by the processing unit 503. Is provided.

  The storage unit 504 collects cDNA sequence data 202 that collects a large number of cDNA sequences, genomic sequence data 203, end information 207 of cDNA sequences that collect information about the sequence ends of each cDNA sequence, and information about the expression of each cDNA sequence. The expression information 209 of the cDNA sequence and the sequence annotation information 212 that collects information on the annotation of each cDNA sequence are stored.

  The processing unit 503 is a mapping processing unit 511 that maps each cDNA sequence of the cDNA sequence data 202 to the genome sequence indicated by the genomic sequence data 203, and a clustering processing unit 512 that forms a cluster of cDNA sequences based on the mapping result. , A variant type classification processing unit 513 that combines cDN sequences having the same sprite site combination as one variant type, a transcription start position classification processing unit 514 that collects cDNA sequences having the same transcription start position, and a cDNA sequence belonging to each variant type Expression information classification processing unit 515 for collecting and classifying expression information, analysis by expression type classification processing unit 516 for each transcription start position for collecting and classifying expression information of cDNA belonging to each transcription start position, and processing unit 503 The result is displayed on the display screen of the display unit 502, for example, as shown in FIG. The display processing unit 517 is provided.

  A processing procedure for obtaining the display shown in FIG. 1 will be described with reference to FIG. In 201, a known genome mapping technique (Non-patent Document 4) is used to map each cDNA sequence to a genome sequence from the cDNA sequence data 202 and the genome sequence data 203 input from the storage unit 504 to the processing unit 503. I do. This processing is performed by the mapping processing unit 511. As a result, it is determined which position on the genome sequence each cDNA sequence is transcribed from. Therefore, the clustering processing unit 512 performs clustering processing 204 of the cDNA sequence by composing a cluster (corresponding to a gene region) by collecting all the cDNA sequences that share one base or several bases in the genome. Do. In order to efficiently perform this clustering process, the coordinates on the genome sequence of both end points of each exon constituting the cDNA sequence are obtained, the genome sequence coordinates are sorted, and the exons including the coordinates, Furthermore, the cDNA sequence containing it may be put in the same cluster.

  Moreover, since the exon / intron structure of each cDNA sequence is determined based on the result of the mapping process 201, the position of the splice site on the genome is determined. The variant type classification processing unit 513 performs variant type classification processing 205 of cDNA sequences by collecting cDNA sequences having the same combination of splice sites as one variant type. Generally, each cDNA sequence has a plurality of splice sites, but when all the splice sites have the same coordinates on the genome sequence or within a specified difference of about several bases or less, the combination of splice sites is the same. . In addition, cDNA sequences having no splice site are collectively classified as one type.

  In addition, due to differences in the procedure when sequencing, the cDNA sequence was (1) sequenced over the entire length, (2) sequenced only on the 5 ′ end, and (3) only on the 3 ′ end. Sequencing, (4) Sequencing of any fragment, (1) and (2) have a complete 5 'end, (1) and (3) Has a complete 3 'end. The transcription start position classification processing unit 514 reads the end point information 207 of the cDNA sequence, which is a collection of information about such sequence ends. When the cDNA sequence has a complete 5 'end and the 5' end is mapped onto the genome sequence in the mapping process 201, the transcription start position is determined for the cDNA sequence. In 206, the transcription start position classification processing unit 514 collects cDNA sequences for which the transcription start position is determined, and classifies them into a group having the same transcription start position. Here, the same transcription start position is assumed to be completely coincident with the genome sequence coordinates, or to be within the difference of the number of bases designated in advance.

  Each cDNA sequence has a variety of information on the original mRNA collected from which site in what organ / tissue of which individual under what conditions. This is called cDNA sequence expression information 209. In the expression information classification processing 208 by variant type by the expression information classification processing unit 515 by variant type, for each variant type obtained in 205, the expression information of cDNA sequences belonging to that type is collected from 209, and these are collected. Classify according to the point of view to be examined. For example, classification according to tissue, classification based on whether it is derived from normal cells, cancer cells, or other diseases, classification based on differences in developmental stages such as fetuses and adults, and external administration such as drug administration Categorize by the presence or absence of stimulation and the difference in elapsed time thereafter. In the expression information classification processing 210 for each transcription start position by the expression information classification processing unit 516 for each transcription start position, for each transcription start position obtained in 206, expression information of cDNA sequences belonging to that position is collected from 209. They are classified in the same way as 208 according to the viewpoint of examining them.

  In a display / GUI (Graphic User Interface) process 211 by the display processing unit 517, a known splice variant comparison display method (Patent Document 1) is applied to one gene region (cluster) selected by the user in FIG. The display shown in 101 is performed. However, cDNA sequences belonging to one variant type are displayed using the most extended end points because there are differences at both ends of the sequences even if the splice sites are common. Further, the classification result of the expression information obtained in 208 for each type is displayed as indicated by 110 in 107 according to 103 coordinates. When the classification result is digitized along the coordinates 108 indicating the expression intensity, for example, the number of EST sequences belonging to the classification is used. However, if the expression information is biased with respect to all EST sequences, in order to correct the bias, the number of EST sequences belonging to one class of one cluster is the number of EST sequences belonging to that class among all EST sequences. The expression intensity is normalized by the number of. Similarly, the classification result of the expression information obtained in 210 for each transcription start position is displayed as indicated by 113 in 111 according to the coordinates of 102.

  In the display / GUI processing 211, the sequence annotation information 212 is read, the annotation information of the sequences belonging to each cluster is collectively displayed in the list box 114, and support information for the user to select a gene region (cluster). And

Hereinafter, a second embodiment of the present invention will be described with reference to the drawings.
FIG. 3 shows an example of displaying the result of splicing variant analysis according to the present invention for a large amount of cDNA sequences.

  3, reference numerals 101 to 113 are the same as those in FIG. 301 is a list box for selecting a cDNA sequence displayed in 101. In each element of the list, an array name indicated by 302 and array annotation information indicated by 303 are displayed. 304 is a marker indicating the element of the currently selected list box. The list box 301 displays cDNA sequences that have been narrowed down, rearranged, or selected according to the conditions described in the search window indicated by reference numeral 305. The selection of 304 is determined by looking at the contents of the list box 301 displayed as a result of the user's input to the search window 305 and reselecting the items in the list box as necessary. 306 and 307 are markers indicating the variant type to which the cDNA sequence selected in 304 belongs, and at the same time indicating their transcription start position and transcription termination position. 308 is a marker indicating the expression information of the variant type to which the cDNA sequence selected in 304 belongs in the window 107 showing the expression information by type. 309 is a marker indicating the expression information at the transcription start position of the cDNA sequence selected at 304 in the window 111 representing the expression information for each transcription start position.

Hereinafter, a third embodiment of the present invention will be described with reference to the drawings.
FIG. 4 shows an example of displaying the result of splice variant analysis according to the present invention for a large amount of cDNA sequences. 101, 103, 106 to 113 are the same as those in FIG. 401 is a box representing one exon included in one type. One type of exon-intron structure is represented by arranging several 401 boxes representing exons along genome sequence coordinates 402. A broken line 403 connecting the boxes 401 represents an intron. However, a box representing an exon at both ends of one type represents the most extended exon belonging to that type on that end point side. This is because the end points of cDNA sequences belonging to one type are different even if the splice site is common.

  As described above, the present invention can be applied to diagnosis for diseases accompanied by abnormal splice variants, evaluation of drug efficacy / toxicity for drugs causing abnormal splice variants, and the like.

The figure which shows the method of displaying collectively the classification result as a splice variant with respect to a large amount of cDNA sequence, the expression information according to classification, and the expression information for every transcription start position. The figure which shows the calculation procedure for performing the display shown in FIG. The figure which shows the 2nd method of displaying collectively the classification result as a splice variant with respect to a lot of cDNA sequences, the expression information according to classification, and the expression information for every transcription start position. The figure which shows the 3rd method of displaying collectively the classification result as a splice variant with respect to a large amount of cDNA sequence, the expression information according to classification, and the expression information for every transcription start position. The schematic block diagram of the splice variant analysis and display apparatus by invention.

Explanation of symbols

102: Genome sequence coordinates
104: A line representing an exon
107: Expression information for each type
111: Expression information for each transcription start position
114: List box for selecting gene regions
118: Search window
301: List box
305: Search window
401: Box representing an exon
403: Line representing intron

Claims (2)

A method for displaying a splice variant by a processing unit that reads and processes a genomic sequence, a plurality of cDNA sequences, and expression information of each cDNA sequence from a storage unit,
The processing unit is
Mapping each of the plurality of cDNA sequences read from the storage unit to the genome sequence read from the storage unit;
A clustering step of classifying cDNA sequences sharing the same base on the genome sequence into one cluster based on information of transcription positions on the genome sequence of each cDNA determined by the mapping process;
A step of classifying cDNA sequences having the same splicing site coordinates on the genome sequence among the cDNAs classified into the clusters or having a difference in coordinates within several bases into one variant type;
Classifying the cDNA sequence classified into the variant type based on expression information;
A step of displaying the display of the exon and intron structure for each variant type of the cluster and the breakdown of the expression information regarding the cDNA sequences classified into each variant type on the display unit,
A method of displaying a splice variant, characterized in that: A method for displaying a splice variant by a processing unit that reads and processes a genomic sequence, a plurality of cDNA sequences, expression information of each cDNA sequence, and information on the sequence end of each cDNA sequence from a storage unit,
The processing unit is
Mapping each of the plurality of cDNA sequences read from the storage unit to the genome sequence read from the storage unit;
A clustering step of classifying cDNA sequences sharing the same base on the genome sequence into one cluster based on information of transcription positions on the genome sequence of each cDNA determined by the mapping process;
A step of classifying cDNA sequences having the same splicing site coordinates on the genome sequence among the cDNAs classified into the clusters or having a difference in coordinates within several bases into one variant type,
Classifying the cDNA sequence classified into the variant type based on expression information;
Transcription of cDNA sequences that are determined based on the result of the mapping process and information on the end of the sequence of the cDNA sequence, the coordinates of the transcription start position on the genome sequence are the same, or the difference in coordinates is within several bases. Categorizing into start position,
Classifying the cDNA sequence classified into the transcription start position based on expression information;
The display of the exon / intron structure for each variant type of the cluster and the breakdown of the expression information regarding the cDNA sequences classified into each variant type are displayed on the display unit in a one-to-one correspondence, and the exon / intron structure is displayed. A step of displaying a breakdown of expression information on the cDNA sequence classified at each transcription start position on the display unit along the genome sequence coordinates used when displaying,
A method of displaying a splice variant, characterized in that:

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