Maeda140303

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1 2014 NGS NIBB FASTA / FASTQ - BED GFF/GTF WIG - SAM / BAM - SAMtools

2 Web HTML (PC/), OS (Windows/Mac), IE/Chrome/Safari

3 NGS Wet - -

4 NGS - FASTA, FASTQ, csfastq, FASTA/qual, SRA, - BED, GFF/GTF, WIG ( - SAM, BAM, PSL, MAF

5 FASTA DB > >gi Bos taurus crystallin, gamma S (CRYGS), mrna TGCACCAAACATGTCTAAAGCTGGAACCAAAATTACTTTCTTTGAAGACAAAA ACTTTCAAGGCCGCCACTATGACAGCGATTGCGACTGTGCAGATTTCCACATG TACCTGAGCCGCTGCAACTCCATCAGAGTGGAAGGAGGCACCTGGGCTGTGTA TGAAAGGCCCA >gi Bos taurus crystallin, gamma S (CRYGS), mrna TGCACCAAACATGTCTAAAGCTGGAACCAAAATTACTTTCTTTGAAGACAAAA ACTTTCAAGGCCGCCACTATGACAGCGATTGCGACTGTGCAGATTTCCACATG TACCTGAGCCGCTGCAACTCCATCAGAGTGGAAGGAGGCACCTGGGCTGTGTA FASTQ NGS (Quality value) 1 ID GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT + ''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65

FASTQ Quality value (Phred quality score) ABI Sequence Scanner QV= -10log10p ( p : ) QV = 30 p = 0.001 QV = 20 p = 0.

6 FASTQ Quality value (Phred quality score) ABI Sequence Scanner QV= -10log10p ( p : ) QV = 30 p = QV = 20 p = 0.01 GATTTGGGGTTCAAAGCAGTATCGATCAAATAGTAAATCCATTTGTTCAACTCACAGTTT + ''*((((***+))%%%++)(%%%%).1***-+*''))**55CCF>>>>>>CCCCCCC65 ASCII QV ASCII: American Standard Code for Information Interchange 1965) 126 A 65 Apple 65;112;112;108;101; FASTQ ASCIIQV( 65 A 10 ( G A T T G G T G A A T A > = ; , QV

ASCII0-32 ASCII 0 null 1 SOH 2 STX p = 0.001 QV=30 NGS10 30 Phred(QV)X ASCII 3 ETX 4 EOT.

7 ASCII0-32 ASCII 0 null 1 SOH 2 STX p = QV=30 NGS10 30 Phred(QV)X ASCII 3 ETX 4 EOT RS XX=33 QV = 63? 31 US " GATTGGTGAATT +??@A>=;9740, ASCII QV33 ASCII

8 @SEQ_ID GATTGGTGAATT p0.01 ASCII33 GATTGGTGAATT p0.01 QV = -10 log10p = -10 log = -10 (-2) = 20 QV < 20 ASCII33 QV G A T T G G T G A A T A > = ; , ASCII63;63;64;65;62;58;59;57;55;52;48;44; QV30;30;31;32;29;25;26;24;22;19;15;11; QV33 ASCII ASCII 33 QV

9 QV Solexa/Illumina Phred(QV)X ASCII ver. (CASAVA) QV Solexa Phred Phred Phred X QV range (2=end of read) 040 FASTQ ( 4 ASCII QV33 ASCII

10 NGS - FASTA, FASTQ, csfastq, FASTA/qual, SRA, - BED, GFF/GTF, WIG ( - SAM, BAM, PSL, MAF BED, GFF/GTF, WIG DB BED GFF GTF GFF

11 BED (Browser Extensible Data format) / Scaffol d 3-12 /,, (0-255) (exon chr clonea ,0, ,488, 0,3512 chr cloneb ,0, ,399, 0,3601 GFF (General Feature Format / Gene Finding Format) 5-9 / Scaffold -. chr22 Manual exon chr22 Manual exon NAME pol1 ; -

12 GTF (General Transfer Format) / Scaffold GFF chr22 Twinscan CDS gene_id 001 ; transcript_id ; chr22 Twinscan CDS gene_id 001 ; transcript_id ; chr22 Twinscan CDS gene_id 001 ; transcript_id ; chr22 Twinscan start_codon gene_id 001 ; transcript_id ; chr22 Twinscan stop_codon gene_id 001 ; transcript_id ; CDS, start_codon, stop_codon 5UTR, 3UTR, inter, inter CNS, intron_cns, exon ID GFF/GTFBED GFF/GTF 1-based (1 BED based, 1-based GFF/GTF A G T A C T C G BED GFF/GTF format : 3, 6 (6-3+1=4) BED format : 2, 6 (6-2=4)

UCSC genome bioinformaticsbed, GTF WIG (Wiggle Format) GC VariableStep variablestep chrom=chr2

13 UCSC genome bioinformaticsbed, GTF WIG (Wiggle Format) GC VariableStep variablestep chrom=chr FixedStep fixedstepchrom=chr3 start= step=

14 NGS - FASTA, FASTQ, csfastq, FASTA/qual, SRA, - BED, GFF/GTF, WIG ( - SAM, BAM, PSL, MAF SAM (Sequence Algnment/Map format) (,, ), VN:1.5 SN:ref LN:45 r ref M2I4M1D3M = TTAGATAAAGGATACTG * r002 0 ref S6M1P1i4M * 0 0 AAAGATAAGGATAT * r003 0 ref S6M * 0 0 GCCTAAGCTAA * SA:Z:ref,29,-,6H5M r004 0 ref M14N5M * 0 0 ATAGCTTCAGC * r ref H5M * 0 0 TAGGC * SA:Z:ref,9,+,5S6M r ref M = 7-39 CAGCGGCAT * NM:i:1

15 @HD VN:1.5 SN:ref VN: ( バージョン )SO: ( ソート状況 SN: ( リファレンス名 ) LN: ( リファレンスの長さ ) FLAG QV CIGAR Ref Q V r ref M2I4M1D3M = TTAGATAAAGGATACTG * r002 0 ref S6M1P1i4M * 0 0 AAAGATAAGGATAT * r003 0 ref S6M * 0 0 GCCTAAGCTAA * SA:Z:ref r004 0 ref M14N5M * 0 0 ATAGCTTCAGC *,29,-, r ref H5M * 0 0 TAGGC * SA:Z:ref r ref M = 7-39 CAGCGGCAT *,9,+, NM:i:1 CIGAR FLAG SAMCIGAR FLAG QV CIGAR r ref M2D2M = GCAAG 44>>> Ref QV 3M2D2M 3 塩基一致 2 個挿入 2 塩基一致 ref ATGCGCATTAGCCTAA read GCA--AG M I D N S H P (RNAvsDNA)

16 SAM FLAG Multiple hit 512 QV, + 10 SAM FLAG Multiple hit 512 QV, + 10

SAM FLAG 10 1 2 4 8 16 32 64 128 256 Multiple hit 16 512 QV 1032 +, 128 160 + 1620 + 80 A0

17 SAM FLAG Multiple hit QV , A0 (16100 Dec 5 Dec Multiple hit QV

18 Dec 5 Dec 419 Hex Hex = 5 ペアリードがある両方マップされていない, 1A3 1 桁目 :1+2 = 3 ペアリードがある両方マップされている 2 桁目 :2+8 = A ペア相手は逆鎖にマップされたペアリードの2 番目である 3 桁目 :1 Multiple hitでトップヒットでないアライメント Multiple hit QV

19 SAM FLAG QV CIGAR r ref M2D2M = GCAAG 44>>> Ref QV FLAG CIGAR + MD (CIGAR -SAM BAM SAM SAM BAM indexing file BAM

20 FASTA FASTQ SAM NGS, DB SNP QVASCII CIGAR, FLAG BAM BED GFF GTF WIG DB GFF GC 2 VariableStep/FixedStep

21 NGS FASTQ BED GFF/GTF SAM/BAM : FASTX-Toolkit, cutadapt, fastqc : BEDtools : SAMtools, Picard SAMtools SAMtools 1-1SAM ( BAM

22 SAMtools SAMtools samtoolscommand Program: samtools (Tools for alignments in the SAM format) Version: cd Usage: Command: view sort mpileup depth faidx tview index idxstats fixmate flagstat calmd merge rmdup reheader cat bedcov targetcut phase bamshuf samtools <command> [options] SAM BAM SAM<->BAM conversion sort alignment file multi-way pileup compute the depth index/extract FASTA text alignment viewer index alignment BAM index stats (r595 or later) fix mate information simple stats recalculate MD/NM tags and '=' bases merge sorted alignments remove PCR duplicates replace BAM header concatenate BAMs read depth per BED region cut fosmid regions (for fosmid pool only) phase heterozygotes shuffle and group alignments by name command 1-1, SAM ( BAM Usage: samtools view [options] <in.bam> <in.sam> [region1 [...]] Options: -b output BAM -h print header for the SAM output -H print header only (no alignments) -S input is SAM -u uncompressed BAM output (force -b) -1 fast compression (force -b) -x output FLAG in HEX (samtools-c specific) -X output FLAG in string (samtools-c specific) -c print only the count of matching records -B collapse the backward CIGAR operation -@ INT number of BAM compression threads [0] -L FILE output alignments overlapping the input BED FILE [null] -t FILE list of reference names and lengths (force -S) [null] -T FILE reference sequence file (force -S) [null] -o FILE output file name [stdout] -R FILE list of read groups to be outputted [null] -f INT required flag, 0 for unset [0] -F INT filtering flag, 0 for unset [0] -q INT minimum mapping quality [0] -l STR only output reads in library STR [null] -r STR only output reads in read group STR [null] -s FLOAT fraction of templates to subsample; integer part as seed [-1] -? longer help sam bam samtools view -S -b./in.sam >./out.bam -S sam -b bam

23 1-1, SAM ( BAM lessex1_7.bam Ex1_7.bam" may be a binary file. See it anyway? y (yes samtools viewex1_7.bamsam samtools view Ex1_7.bam (bam sam ls Ex1_7.sam, Ex1_7.bam ls -l ls = -l = long sam BAMHDD SAM 1-2 Step 1 Step 2 samtools sort samtools index Usage: samtools sort [-on] [-m <maxmem>] <in.bam> <out.prefix> input bam out Usage: samtools index <in.bam> [out.index]

sam Ex1_7.sam ) head -n 10 Ex1_7.sam head -n 10 Ex1_7_s.

24 1-2 Step 1 samtools sort samtools viewex1_7_s.bamsam samtools view Ex1_7_s.bam > Ex1_7_s.sam head tail Ex1_7_s.sam Ex1_7.sam ) head -n 10 Ex1_7.sam head -n 10 Ex1_7_s.sam tail -n 10 Ex1_7.sam tail -n 10 Ex1_7_s.sam samtools sort Ex1_7.bam Ex1_7_s Ex1_7_s.sam 1-2 Step 2 samtools index

25 SAMtools 1-1SAM ( BAM , 2-1 samtools view indexview samtools view Ex1_7_s.bam cp1: samtools view -f/-f (flag -f/-f -f flag -F flag samtools view -f 83 Ex1_sort.bam

26 2, 2-1 rbcl rpoc samtools view Ex1_7_s.bam cp1: wc -l Multiple hit 512 QV 2, 2-2 samtools flagstat samtools idxstatsref samtools depth

2, 2-2 samtools flagstat Usage: samtools flagstat <in.bam> 200000 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 duplicates 48481 + 0 mapped (24.

27 2, 2-2 samtools flagstat Usage: samtools flagstat <in.bam> in total (QC-passed reads + QC-failed reads) duplicates mapped (24.24%:nan%) paired in sequencing read read properly paired (23.52%:nan%) with itself and mate mapped singletons (0.32%:nan%) with mate mapped to a different chr with mate mapped to a different chr (mapq>=5) 2, 2-2 samtools idxstatsref Usage: samtools idxstats <in.bam> cp * samtools depth Usage: samtools depth [options] in1.bam [in2.bam [...]] Cp Cp Cp Cp

28 SAMtools samtools view sort index flagstat idxstats depth SAMtools mpileup SNP tview merge BAM rmdup PCR duplicate

<4D F736F F F696E74202D D D E C815B836A F B83582E >

<4D F736F F F696E74202D D D E C815B836A F B83582E > 2012.03.22 マッピングデータの基本フォーマットと基本ツール次世代シーケンサーに良く用いられるファイル形式 Samtools Integrative Genomics Viewer(IGV) 基礎生物学研究所生物機能解析センター山口勝司 NIBB CORE RESEARCH FACILITIES FUNCTIONAL GENOMICS FACILITY NIBB CORE RESEARCH