お家でできるMacBookでやる次世代シーケンスデータ解析 pdf

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1 Mac Book

2 @n0rr 2012/03/ /03/30

3 R UNIX UNIX/ Linux Mac Book Pro Mac UNIX NGS SEQanswers Mac Book ii

4 1 UNIX bowtie

5 1 Bowtie UNIX Linux 1. Bowtie 2. Bowtie 3. Bowtie Mac Mac Book Pro Apple Store Lion Mac OS X GHz Intel nor mac Core i5 8G 1333MHz DDR3 Macintosh HD 13-inch HDD 320GB HDD 4 4

6 Dock ls Last login: Sat Jan 21 18:07:52 on ttys000 dhcp :~ nor$ ls Desktop Documents Downloads Library Movies Music Pictures Public R analtools dhcp :~ nor$ Finder [ ] [ ] [ ] [ ] [Pro] Last login: Sat Jan 21 18:07:52 on ttys000 dhcp :~ nor$ nor nor Mac Book Pro /Users nor ls -a 5

7 dhcp :~ nor$ ls -a....cfusertextencoding.ds_store.rdata.rapp.history -l dhcp :~ nor$ ls -l total 2944 drwx nor staff :16 Desktop drwx nor staff :53 Documents drwx nor staff :14 Downloads Bowtie analtools dhcp :~ nor$ mkdir analtools /Users/nor/ /Users/ nor/analtools Bowtie Bowtie bowtie-bio/files/bowtie/0.12.7/bowtie macos-10.5-x86 _64.zip/download sourceforge 7/ bowtie macos-10.5-x86_64.zip analtools cd analtools ls dhcp :~ nor$ cd analtools dhcp :analtools nor$ ls bowtie macos-10.5-x86_64.zip bowtie macos-10.5-x86_64.zip unzip dhcp :analtools nor$ unzip bowtie macos-10.5-x86_64.zip bowtie macos-10.5-x86_64.zip / Users/nor/analtools bowtie ls cd bowtie

8 dhcp :downloads nor$ cd bowtie dhcp :bowtie nor$ Bowtie make Bowtie dhcp :bowtie nor$ /Users/nor/analtools/bowtie /bowtie No index, query, or output file specified! Usage: bowtie [options]* <ebwt> {-1 <m1> -2 <m2> --12 <r> <s>} [<hit>] Bowtie tab /Users/nor/analtools/bowtie /reads dhcp :bowtie nor$ cd reads/ dhcp :reads nor$ ls e_coli_1000.fa e_coli_10000snp.fa e_coli_1000_1.fq e_coli_1000.fq e_coli_10000snp.fq e_coli_1000_2.fa e_coli_1000.raw e_coli_1000_1.fa e_coli_1000_2.fq Genome Analyzer Ⅱx fastq.fq e_coli_1000.fq more dhcp :reads nor$ more GAACGATACCCACCCAACTATCGCCATTCCAGCAT + CCGAACTGGATGTCTCATGGGATAAAAATCATCCG + TCAAAATTGTTATAGTATAACACTGTTGCTTTATG + EDCCCBAAAA@@@@?>===<;;9: more GAACGATACCCACCCAACTATCGCCATTCCAGCAT + EDCCCBAAAA@@@@?>===<;;9: ID + + ID 7

9 +ID more [control] [c] wc -l e_coli_1000.fq 1000 /Users/nor/analtools/bowtie /genomes fastq dhcp :reads nor$ more e_coli_1000.fq wc -l 4000 wc UNIX more e_coli_1000.fq wc dhcp :reads nor$ cd.. dhcp :bowtie nor$ cd genomes/ dhcp :genomes nor$ ls NC_ fna cd cd.. /Users/nor/analtools/bowtie /genome cd.. /Users/nor/analtools/bowtie / cd genomes/ /Users/nor/analtools/bowtie /genomes cd../genomes/ ls NC_ fna.fna fasta more wc 8

10 dhcp :genomes nor$ more NC_ fna >gi ref NC_ Escherichia coli 536, complete genome AGCTTTTCATTCTGACTGCAACGGGCAATATGTCTCTGTGTGGATTAAAAAAAGA GTGTCTGATAGCAGCTTCTGAACTGGTTACCTGCCGTGAGTAAATTAAAATTTTA TTGACTTAGGTCACTAAATACTTTAACCAATATAGGCATAGCGCACAGACAGATA AAAATTACAGAGTACACAACATCCATGAAACGCATTAGCACCACC dhcp :genomes nor$ more NC_ fna wc k bowtie dhcp :genomes nor$ /Users/nor/analtools/bowtie /bowtie-build NC_ fna NC_ bowtie-build bowtie-build Path NC_ fna NC_ Path ls cd wc /usr/bin/ Path bowtie-build Path /Users/nor/analtools/ bowtie / Path / use/bin install install Path dhcp :genomes nor$ PATH=$PATH:/Users/nor/analtools/bowtie / bowtiebuild ls dhcp :genomes nor$ ls NC_ ebwt NC_ ebwt NC_ rev.2.ebwt NC_ ebwt NC_ fna NC_ ebwt NC_ rev.1.ebwt dhcp :genomes nor$ /Users/nor/analtools/bowtie /bowtie -l 32 -n 2 --best -p 1 /Users/nor/analtools/bowtie /genomes/NC_ /Users/nor/analtools/bowtie /reads/e_coli_1000.fq more /Users/nor/ analtools/bowtie /bowtie bowtie Path bowtie -l 32 bowtie 32 -n 2 SNP 9

11 --best -p 1 4 Path Path bowtie more ID dhcp :genomes nor$ /Users/nor/analtools/bowtie /bowtie -l 32 -n 2 --best -p 1 /Users/nor/analtools/bowtie /genomes/NC_ /Users/nor/analtools/bowtie /reads/e_coli_1000.fq > bowtie_mapped_e_coli_1000_2_nc_ txt > bowtie_mapped_e_coli_1000_2_nc_ txt /Users/nor/analtools/bowtie /genomes /Users/nor/ Desktop dhcp :genomes nor$ /Users/nor/analtools/bowtie /bowtie -l 32 -n 2 --best -p 1 /Users/nor/analtools/bowtie /genomes/NC_ /Users/nor/analtools/bowtie /reads/e_coli_1000.fq > /Users/nor/Desktop/bowtie_mapped_e_coli_1000_2_NC_ txt # reads processed: 1000 # reads with at least one reported alignment: 697 (69.70%) # reads that failed to align: 303 (30.30%) Reported 697 alignments to 1 output stream(s) bowite analtools (ls, mkdir) 3. Bowtie analtools 10

12 4. analtools (cd) 5. Bowtie (unzip) 6. (ls, cd, more, wc) 7. (ls, cd, more, wc) 8. (bowtie-build) 9. (bowtie) 11

13 2 Bowtie Bowtie 1. Bowtie 2. DDBJ 3. DDBJ Sequence Read Archive [ ] DRASearch Kc167 mrna SRR DRASearch Accession SRR Search 12

14 SRR Kc167 Single Pair end ) Trizol Illumina GA 36bp SRR fastq.bz2 /Users/nor/Download/ Finder dhcp :~ nor$ mkdir drkc167 dhcp :~ nor$ cd drkc167 /Users/nor/ /Users/nor/drkc167/ drkc167 Finder FASTQ SRR fastq.bz2 FTP SRR fastq.bz2 DRASearch Study SRR fastq.bz2 /Users/nor/drkc167 dhcp :~ drkc167$ cp /Users/nor/Download/SRR fastq.bz2 /Users/nor/drkc167/SRR fastq.bz2 ls SRR fastq.bz2 /Users/ nor/drkc167/.fastq.bz2 bzip2 fastq 13

15 dhcp :drkc167 nor$ bzip2 -dc SRR fastq.bz2 > SRR fastq > fastq ls dhcp :drkc167 nor$ ls -l total rw-r--r-- 1 nor staff :36 SRR fastq -rw-r--r--@ 1 nor staff :34 SRR fastq.bz2 more fastq wc dhcp :drkc167 nor$ wc -l SRR fastq SRR fastq SRR fastq ID SRR HWI-EAS440_42_2_1_N_N / Ensemble cdna dhcp :drkc167 nor$ more HWI-EAS440_42_2_1_1600_2041 ACCGAAGCCATACGAGAGGCCCGCGGACTTCATCGA + HWI-EAS440_42_2_1_785_608 ACGAGTTCCAGAACTTGCACGCCGTTGCCAAGGAGA + HWI-EAS440_42_2_1_1408_814 ATAAAGTTCATGGCATTTGAAGAAAAACTGTGTTTA + IIIIII+IIIIIIIIIIIIII1IIIIIIIIIIIIII Drosophila melanogaster cdna [FASTA] FTP 14

16 genomic DNA [README] all ab initio README all cdna ab initio cdna all /Users/ nor/download/ / Users/nor/drkc167/ n0rr:drkc167 nor$ gunzip Drosophila_melanogaster.BDGP5.65.cdna.all.fa.gz n0rr:drkc167 nor$ /Users/nor/analtools/bowtie /bowtie -l 36 -n 2 -p 4 Drosophila_melanogaster.BDGP5.65.cdna.all SRR fastq > bowmapped_l36n2_srr txt /Users/nor/analtools/ bowtie /bowtie bowtie path bowtie -l 36 -n 2 -p 4 CPU > (bowmapped_l36n2_srr txt) bowtie more bowtie n0rr:drkc167 nor$ /Users/nor/analtools/bowtie /bowtie-build Drosophila_melanogaster.BDGP5.65.cdna.all.fa Drosophila_melanogaster.BDGP5.65.cdna.all 15

17 SRR HWI-EAS440_42_2_1_1326_1394 ID + FBtr AGCGCATCCGCGTCAAGCTCGACGGCTCCCAGCTGG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII :G>C G C bowtie # reads processed: # reads with at least one reported alignment: (84.59%) # reads that failed to align: (15.41%) Reported alignments to 1 output stream(s) phix 98.5% 80% mrna 50-60% unix n0rr:drkc167 nor$ more bowmapped_l36n2_srr txt awk -F "\t" '{print $3}' sort uniq -c awk '{print $2"\t"$1}' > count_bowmapped_l36n2_srr txt 16

18 more [control] [c] awk awk unix -F \t \t {print } {print $3} sort uniq -c awk $1 $2 A, B, C n0rr:drkc167 nor$ more ABC.txt A B C A B A A B B C n0rr:drkc167 nor$ more ABC.txt sort uniq A B C n0rr:drkc167 nor$ more ABC.txt sort uniq -c 2 A 2 B 1 C awk n0rr:drkc167 nor$ more ABC.txt sort uniq -c awk '{print $2"\t"$1}' A 2 B 2 C 1 n0rr:drkc167 nor$ more ABC.txt sort more 17

19 cdna n0rr:drkc167 nor$ more count_bowmapped_l36n2_srr txt 1 FBtr FBtr FBtr FBtr FBtr FBtr FBtr wc cdna 1. (mkdir) (cp) 4. (bzip2 -dc) 5. (more, wc -l) (gunzip) 9. (bowtie-build) 10. (bowtie) n0rr:drkc167 nor$ more Drosophila_melanogaster.BDGP5.65.cdna.all.fa grep ">" wc -l /25415=74% 11. (more) 12. (more, awk, sort, uniq -c) mrna 18

20 3 bowtie cdna bowtie -n SNP Indel SNP Indel SNP Indel bowtie SNP Indel Indel bwa bwa 19

21 HapMap ID H_IJ-NA18973 DRA Experiment: Study: 6 Run: Run.fastq.bz2 HP DNA SRR077861_1.fastq.bz2 SRR077861_2.fastq.bz2 SRR fastq.bz2 DNA DNA cdna acc=erx /Users/nor/ dhcp :~ nor$ mkdir homo_snp_srr

22 bzip2 dc dhcp :~ homo_snp_srr077861$ bzip2 -dc SRR077861_1.fastq.bz2 > SRR07786_1.fastq dhcp :~ homo_snp_srr077861$ bzip2 -dc SRR077861_2.fastq.bz2 > SRR07786_2.fastq 13 ftp://genome-ftp.cse.ucsc.edu/goldenpath/hg19/bigzips/ chromfa.tar.gz dhcp :~ homo_snp_srr077861$ tar zxvf chromfa.tar.gz cat dhcp :~ homo_snp_srr077861$ cat *.fa > chr_all.fa.fa dhcp :~ homo_snp_srr nor $ mkdir reference dhcp :~ homo_snp_srr nor $ mv chr_all.fa./reference/chr_all.fa bwa bwa source forge /Users/nor/analtools/ /Users/nor/ analtools/ bwa / Users/nor/analtools/bwa/ bwa tar.bz2 n0rr:nakagawa nor$ cd /Users/nor/analtools/bwa/ n0rr:bwa nor$ tar zxvf bwa tar.bz2 /Users/nor/analtools/bwa/bwa-0.6.1/ n0rr:bwa nor$ cd bwa n0rr:bwa nor$ make 21

23 make bwa make install bwa /usr/bin/ path norr:bwa nor$ make -bash: make: command not found xcode Snow Leopard Lion xcode 3G sdk MacOSx10.5 n0rr:bwa nor$ /Users/nor/analtools/bwa/bwa-0.6.1/bwa Program: bwa (alignment via Burrows-Wheeler transformation) Version: r104 Contact: Heng Li Usage: bwa <command> [options] Command: index index sequences in the FASTA format aln gapped/ungapped alignment samse generate alignment (single ended) sampe generate alignment (paired ended) bwasw BWA-SW for long queries fastmap identify super-maximal exact matches fa2pac convert FASTA to PAC format pac2bwt generate BWT from PAC pac2bwtgen alternative algorithm for generating BWT bwtupdate update.bwt to the new format bwt2sa generate SA from BWT and Occ pac2cspac convert PAC to color-space PAC stdsw standard SW/NW alignment bwa bwa bowtie bwa path n0rr:bwa nor$ PATH=$PATH:/Users/nor/analtools/bwa/bwa-0.6.1/ bwa path bwa n0rr:bwa nor$ bwa 22

24 /Users/ nor/homo_snp_srr077861/reference /Users/nor/homo_SNP_SRR bwa dhcp :homo_snp_srr nor$ /Users/nor/analtools/bwa/bwa-0.5.9/bwa index -a bwtsw./reference/chr_all.fa./ reference/chr_all.fa reference chr_all.fa /Users/nor/homo_SNP_SRR077861/reference/chr_all.fa bwa dhcp :homo_snp_srr nor$ /Users/nor/analtools/bwa/bwa-0.5.9/bwa aln./reference/chr_all.fa SRR077861_1.fastq > aln_sa1.sai dhcp :homo_snp_srr nor$ /Users/nor/analtools/bwa/bwa-0.5.9/bwa aln./reference/chr_all.fa SRR077861_2.fastq > aln_sa2.sai dhcp :homo_snp_srr nor$ /Users/nor/analtools/bwa/bwa-0.5.9/bwa sampe./reference/chr_all.fa aln_sa1.sai aln_sa2.sai SRR077861_1.fastq SRR077861_2.fastq > bwa_aln.sam.sam Sequence Alignment/Map (SAM) samtools bowtie SAM -S samtools sourceforge samtools /Users/nor/analtools samtools samtools 23

25 dhcp :homo_snp_srr nor$ cd /Users/nor/analtools/ dhcp :analtools nor$ mkdir samtools dhcp :analtools nor$ cd samtools samtools tar.bz2 dhcp :analtools nor$ tar zxvf samtools samtools tar.bz2 samtools dhcp :analtools nor$ cd samtools samtools dhcp :analtools nor$ make samtools dhcp :homo_snp_srr nor$ /Users/nor/analtools/samtools/samtools /samtools view -b -o bwa_aln.sam.bam -S -t./reference/chr_all.fa.fai bwa_aln.sam sam samtools view bam bam more dhcp :homo_snp_srr nor$ /Users/nor/analtools/samtools/samtools /samtools sort bwa_aln.sam.bam bwa_aln.sam.bam.sorted bam dhcp :homo_snp_srr nor$ /Users/nor/analtools/samtools/samtools /samtools mpileup -uf./reference/chr_all.fa bwa_aln.sam.bam.sorted.bam /Users/nor/analtools/samtools/samtools /bcftools/bcftools view -bvcg - > bwa_aln.var.raw.bcf samtools mpileup -f bcf varfilter dhcp :homo_snp_srr nor$ /Users/nor/analtools/samtools/samtools /bcftools/bcftools view bwa_aln.var.raw.bcf /Users/nor/analtools/samtools/samtools /bcftools/vcfutils.pl varfilter -D 500> bwa_aln.var.vcf vcf vcf samtools HP 1. (bzip2 -dc/ tar zxvf) 2. (mv) 24

26 3. (make) 4. (bwa) 5. (samtools) 6. (samtools) Mac Book Air E. coli. ftp://ftp.ddbj.nig.ac.jp/ddbj_database/dra/fastq/sra008/s RA008271/SRX SRR fastq.bz2 Send File FASTA Create File sequence.fasta /Users/nor/ Downloas/ /Users/nor/otherPJ/macde/ ecoli otherpj $nor cd /Users/nor/ $nor mkdir otherpj $nor cd otherpj $nor mkdir macde $nor cd macde $nor mkdir ecoli $nor cd ecoli /Users/nor/Download/ (SRR fastq.bz2) 25

27 (sequence.fasta) Finder /Users/nor/otherPJ/macde/ ecoli/ mv nor$ mv /Users/nor/Downloads/SRR fastq.bz2././ nor$ mv /Users/nor/Downloads/sequence.fasta./ samtools fai nor$ /Users/nor/analtools/samtools/samtools /samtools faidx sequence.fasta bam bowtie nor$ /Users/nor/analtools/bowtie /bowtie-build sequence.fasta sequence nor$ bzip2 -cd SRR fastq.bz2 > SRR fastq nor$ /Users/nor/analtools/bowtie /bowtie -n 2 -l 36 -p 4 -S sequence SRR fastq > bowmap_srr sam nor$ /Users/nor/analtools/samtools/samtools /samtools view -b -o aln.sam.bam -S -t sequence.fasta.fai bowmap_srr sam sort nor$ /Users/nor/analtools/samtools/samtools /samtools sort aln.sam.bam aln.sam.bam.sorted nor$ /Users/nor/analtools/samtools/samtools /samtools mpileup -uf sequence.fasta aln.sam.bam.sorted.bam 26

28 /Users/nor/analtools/samtools/samtools /bcftools/bcft ools view -bvcg - > aln.var.raw.bcf varfilter nor$ /Users/nor/analtools/samtools/samtools /bcftools/bcft ools view aln.var.raw.bcf /Users/nor/analtools/samtools/samtools /bcftools/vcf utils.pl varfilter > aln.var.vcf aln.var.vcf PCR duplicate bam nor$ /Users/nor/analtools/samtools/samtools /samtools mpileup -uf sequence.fasta aln.uniq.bam /Users/nor/analtools/samtools/samtools /bcftools/bcft ools view -bvcg - > aln.uniq.var.raw.bcf varfilter $nor /Users/nor/analtools/samtools/samtools /bcftools/bcft ools view aln.uniq.var.raw.bcf /Users/nor/analtools/samtools/samtools /bcftools/vcf utils.pl varfilter > aln.uniq.var.vcf nor$ /Users/nor/analtools/samtools/samtools /samtools mpileup -f sequence.fasta aln.sam.bam.sorted.bam > aln.cov.txt PCR duplicate bam samtools rmdup PCR duplicate nor$ /Users/nor/analtools/samtools/samtools /samtools rmdup -s aln.sam.bam.sorted.bam aln.uniq.bam nor$ /Users/nor/analtools/samtools/samtools /samtools mpileup -f sequence.fasta aln.uniq.bam > aln.uniq.cov.txt 27

29 チャプタ 2 Rをつかって やるところ 発現変動遺伝子抽出 カバレッジの統計をとる

30 1 UNIX R R 1. R 2. R 3. R R /03/ R SRA RNA-seq 29

31 SRR001358, SRR bowtie cdna( ftp://ftp.ensembl.org/pub/release-66/fasta/mus_musculus/cdna/ Mus_musculus.NCBIM37.66.cdna.all.fa.gz) -S.sam awk -F "\t" '{print $3}' sort uniq -c /Users/nor/ / Users/nor/otherPJ/macde/ R join nor$ cat gene_counts_liver.txt gene_counts_skemuscle.txt awk '{print $2}' sort uniq > genename.txt $ more Mus_musculus.NCBIM37.66.cdna.all.fa grep ">" awk -F " " '{print $1}' tr -d ">" sort > genename.txt join $ more gene_counts_liver.txt awk {print $2 \t $1} > temp1 30

32 join sort sort tophat CASAVA sort more filename sort > filename2 nor$ more gene_counts_skemuscle.txt awk '{print $2"\t"$1}' > temp2 join nor$ join a 1 -a 1 genename.txt temp1 awk '{print $1"\t"$2+0}' more temp11 nor$ join a 1 -a 1 genename.txt temp1 awk '{print $1"\t"$2+0}' > temp11 nor$ join a 1 -a 1 genename.txt temp2 awk '{print $1"\t"$2+0}' > temp21 wc temp1 temp2 temp11 temp21 UNIX 31

33 paste awk temp3 nor$ paste temp11 temp21 awk '{print $1"\t"$2"\t"$4}' > temp3 $ vi header.txt vim i INSERT genename liver skmus [esc] [:][w][q] vim header.txt CR mi LF 32

34 temp3 nor$ cat header.txt temp3 > for_r_liv_vs_skm.txt R R data<-read.table("/users/nor/otherpj/macde/for_r_liv_vs_ skm.txt",header=t,row.names=1) data /Users/nor/otherPJ/macde/ 33

35 for_r_liv_vs_skm.txt data attach liver skelm summary liver skelm (Differentially Expressed Genes/ DEG) DESeq DEG DESeq HP source(" plot(log(liver),log(skelm),pch=20) bioclite("deseq", dependencies=true) DESeq DESeq (n=1) out_f <- "DESeq_out_liv_vs_skelm.txt" param1 <- 1 param2 <- 1 library(deseq) data.cl <- c(rep(1, param1), rep(2, param2)) cds <- newcountdataset(data, data.cl) 34

36 cds <- estimatesizefactors(cds) sizefactors(cds) cds <- estimatevariancefunctions(cds, method="blind") out <- nbinomtest(cds, 1, 2) logratio <- out$log2foldchange FDR <- out$padj rank_fdr <- rank(fdr, ties.method="min") tmp <- cbind(rownames(data), data, logratio, FDR, rank_fdr) write.table(tmp, out_f, sep="\t", append=f, quote=f, row.names=f) FDR DEG55 (FDR<0.05) DEG /Users/nor/ DESeq_out_liv_vs_skelm.txt FDR DEG FDR False Discovery Rate p 35

37 par(mfrow=c(1,2)) barplot(matrix(sort(liver/sum(liver)))) barplot(matrix(sort(skelm/sum(skelm))),ylim=c(0,1)) 36

38 2 genomic DNA kc167 DNA genomic DNA SRR DRA 1. maq maq maq user s manual Mapass2 Work Flow maq PATH maq easyrun pileup /Users/nor/analtools/maq-0.7.1/scripts/maq.pl easyrun./dmel_genome/no_space_dmg.fa srr fastq && cd easyrun && maq pileup ref.bfa all.map > all_pileup.txt R R awk '($1=="chr2L"){print $2"\t"$4}' all_pileup.txt > chr2l_pileup_r.txt && 37

39 awk '($1=="chr3L"){print $2"\t"$4}' all_pileup.txt > chr3l_pileup_r.txt && awk '($1=="chr2R"){print $2"\t"$4}' all_pileup.txt > chr2r_pileup_r.txt && awk '($1=="chr3R"){print $2"\t"$4}' all_pileup.txt > chr3r_pileup_r.txt && awk '($1=="chr4"){print $2"\t"$4}' all_pileup.txt > chr4_pileup_r.txt && awk '($1=="chrX"){print $2"\t"$4}' all_pileup.txt > chrx_pileup_r.txt && awk '($1=="chrMT"){print $2"\t"$4}' all_pileup.txt > chrmt_pileup_r.txt R chr3r_pileup_r.txt R pos count plot(pos,count,pch=20,col=ifelse(count < 1, "red", "black")) 38

40 0rate 0 13 vs 10 chr3l kc167 MT chr length Zero 0rate Cov. Max chr3l chrx chrmt chr2l chr2r chr3r chr small RNA 39

41 チャプタ 3 その他の問題 と対処 bcl変換とデマルチプレックス クオリティチェック コントロール 記憶媒体のフォーマット

42 1 bcl illumina Genome Analyzer Ⅱx bcl stats fastq CASAVA fastq 1. CASAVA illumina Genome Analyzer Ⅱx fastq 2. fastq UNIX bash 3. bash bcl Illumina CASAVA CASAVA illumina CASAVA v1.8.0 $ tar -jxf CASAVA_v1.8.0.tar.bz2 $ mkdir CASAVA_v1.8.0-build $ cd CASAVA_v1.8.0-build $../CASAVA_v1.8.0/configure $ make $ make install CASAVA_v1.8.0.tar.bz2 / Users/nor/analtools/ /analtools make install 41

43 bcl Genome Analyzer _HWUSI- EAS1748_00049_FC CASAVA HDD bcl HDD fastq fastq.gz 70bp 10GB fastq 100GB HDD LOGITEC_HD test /Volumes/LOGITEC_HD/test/120114_HWUSI- EAS1748_00049_FC/ SSD NOGEBLANC /Volumes/NOGEBLANC/ _5 nor$ configurebcltofastq.pl --input-dir /Volumes/LOGITEC_HD/test/120114_HWUSI-EAS1748_00049_FC/Data/Inten sities/basecalls/ --output-dir /Volumes/NOGEBLANC/120114_5 --use-bases-mask y72 --use-bases-mask y72 65bp( )+7bp( ) [ :09:49] [configurebcltofastq.pl] INFO: Creating directory '/Volumes/NOGEBLANC/120114_5' [ :09:49] [configurebcltofastq.pl] INFO: Basecalling software: RTA [ :09:49] [configurebcltofastq.pl] INFO: version: 1.09 (build 35) [ :09:49] [configurebcltofastq.pl] WARNING: 'LocationFileType' element not found in /Volumes/LOGITEC_HD/test/120114_HWUSI-EAS1748_00049_FC/Data/Intensities/BaseCalls/../RTAConfiguration.xml [ :09:49] [configurebcltofastq.pl] INFO: Original use-bases mask: y72 [ :09:49] [configurebcltofastq.pl] INFO: Guessed use-bases mask: yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy,iiiiiin Creating directory '/Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane1' Creating directory '/Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane2' Creating directory '/Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane3' Creating directory '/Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane4' Creating directory '/Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane5' Creating directory '/Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane6' Creating directory '/Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane7' Creating directory '/Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane8' [ :09:50] [configurebcltofastq.pl] WARNING: Couldn't determine control software version due to missing /Volumes/LOGITEC_HD/test/120114_HWUSI-EAS1748_00049_FC/Data/Intensities/BaseCalls/../../../runParameters.x ml [ :09:50] [configurebcltofastq.pl] WARNING: use-base-mask length is 72 for read 1: expected length from config file is 65 [ :09:50] [configurebcltofastq.pl] INFO: Read 1: length = 72: mask = yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy 42

44 [ :09:50] [configurebcltofastq.pl] WARNING: Read 2: current cycle in mask is 73: first cycle in read is 66 [ :09:50] [configurebcltofastq.pl] INFO: Read 2: length = 7: mask = IIIIIIn [ :09:50] [configurebcltofastq.pl] WARNING: use-base-mask length is 72 for read 1: expected length from config file is 65 [ :09:50] [configurebcltofastq.pl] WARNING: Read 2: current cycle in mask is 73: first cycle in read is 66 [ :09:50] [configurebcltofastq.pl] INFO: Running self tests: 'make self_test' [ :09:53] [configurebcltofastq.pl] INFO: Running self tests on /Volumes/NOGEBLANC/120114_5 completed with no problems /Volumes/NOGEBLANC/120114_5 make n0rr:120114_4 nor$ cd /Volumes/NOGEBLANC/120114_5/ n0rr:120114_5 nor$ nohup make -j 2 nohup.out configurebcltofastq.pl (Basecalls bcl /Volumes/NOGEBLANC/120114_5 fastq.gz dhcp :120114_5 nor$ cd /Volumes/NOGEBLANC/120114_5/Project_FC/ Sample_lane1/ dhcp :sample_lane1 nor$ gunzip lane1_noindex_l001_r1_001.fastq.gz more wc fastq nor$ more lane1_noindex_l001_r1_001.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/ K@HWUSI/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' > line_lane1.fastq.txt nor$ more line_lane1.fastq.txt grep "ATCACG$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_1_1_index1_atcacg.fastq 43

45 tr 3. [sed -e K tr A B A B sed awk 4. [tr K \n ] K grep more more fasq 1. [tr " " "_ ] 5. [awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}'] + 2. [tr \n \t ] 6. [> line_lane1.fastq.txt] 44

46 fastq grep grep "ATCACG$" ATCACG $ fastq fastq fastq.gz bash.sh chmod sh # $1: lane number. so 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 # This command lines are prepared for 65 bp single reads. If you want to use them for other length reads, modify the number of "substr" region. 65->N and 66->N+1. # Skip the unsupporting index separating greps. Use "#" to inactivation. #making fastq 2 linefastq, separating index reads, and assembling them. more lane"$1"_noindex_l00"$1"_r1_001.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' > line_lane"$1".fastq.txt && more lane"$1"_noindex_l00"$1"_r1_002.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' >> line_lane"$1".fastq.txt && more lane"$1"_noindex_l00"$1"_r1_003.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' >> line_lane"$1".fastq.txt && more lane"$1"_noindex_l00"$1"_r1_004.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' >> line_lane"$1".fastq.txt && more lane"$1"_noindex_l00"$1"_r1_005.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' >> line_lane"$1".fastq.txt && more lane"$1"_noindex_l00"$1"_r1_006.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' >> line_lane"$1".fastq.txt && more lane"$1"_noindex_l00"$1"_r1_007.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' >> line_lane"$1".fastq.txt && more lane"$1"_noindex_l00"$1"_r1_008.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' >> line_lane"$1".fastq.txt && more lane"$1"_noindex_l00"$1"_r1_009.fastq tr " " "_" tr "\n" "\t" sed -e 's/@hwusi/k@hwusi/g' tr "K" "\n" awk -F "\t" '{print $1"\t"substr($2,1,65)"\t+\t"substr($4,1,65)"\t"substr($2,66,6)}' >> line_lane"$1".fastq.txt && 45

47 echo "fastq2linefastq" more line_lane"$1".fastq.txt grep "TAGCTT$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index10_tagctt.fastq && #making fastq by an index. #Index1 more line_lane"$1".fastq.txt grep "ATCACG$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index1_atcacg.fastq && #Index2 #index11 more line_lane"$1".fastq.txt grep "GGCTAC$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index11_ggctac.fastq && #Index12 more line_lane"$1".fastq.txt grep "CTTGTA$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index12_cttgta.fastq && more line_lane"$1".fastq.txt grep "CGATGT$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index2_cgatgt.fastq && #Index3 more line_lane"$1".fastq.txt grep "TTAGGC$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index3_ttaggc.fastq && echo "finish (^o^)b" #Index4 more line_lane"$1".fastq.txt grep "TGACCA$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index4_tgacca.fastq && #Index5 more line_lane"$1".fastq.txt grep "ACAGTG$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index5_acagtg.fastq && #Index6 more line_lane"$1".fastq.txt grep "GCCAAT$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index6_gccaat.fastq && #index7 more line_lane"$1".fastq.txt grep "CAGATC$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index7_cagatc.fastq && #index8 more line_lane"$1".fastq.txt grep "ACTTGA$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index8_acttga.fastq && #index9 more line_lane"$1".fastq.txt grep "GATCAG$" awk '{print $1"\n"$2"\n"$3"\n"$4}' > s_"$1"_1_index9_gatcag.fastq && #index10 split_index_for_mac_unix.sh /Volumes/NOGEBLANC/120114_5/ Project_FC/Sample_lane1/ lane1_noindex_l001_r1_001.fastq, lane1_noindex_l001_r1_002.fastq, lane1_noindex_l001_r1_003.fastq, lane1_noindex_l001_r1_004.fastq /Volumes/NOGEBLANC/ _5/Project_FC/Sample_lane1/ split_index_for_mac_unix.sh 46

48 nor$ cd /Volumes/NOGEBLANC/120114_5/Project_FC/Sample_lane1/ nor$ chmod 777 split_index_for_mac_unix.sh nor$ sh split_index_for_mac_unix.sh 1 $1 # Index 1-12 grep # 47

49 2 fastq Illumina Genome Analyzer Ⅱx HiSeq 1. FastQC FastQC FastQC [Download Now] [FastQC v (Mac DMG image)] Finder 48

50 FastQC [ ] [File] [Open] /Users/nor/ drkc167/ fastq [File] [Save report] zip html zip html 11 Basic Statistics Per base sequence quality Per sequence quality scores Per base sequence content Per base GC content Per sequence GC content 49

51 Per base N content Sequence Length Distribution Sequence Duplication Levels Per base sequence content Overrepresented sequences Kmer Content Basic Statistics Per base GC content GC Per sequence GC content GC AT Per base sequence quality AT GC HiSeq Per base N content N Per sequence quality scores Sequence Length Distribution 50

52 cut adapt Sequence Duplication Levels GenomeJack Sequence Duplication Levels PCR PCR RNA-seq Overrepresented sequences Kmer Content Kmer Kmer 5 GenomeJack bioinformatics/solution/products/genomejack/index.html (@genomejack ) PC 51

53 PCR FastQC 90% PCR Duplication Levels PCR Duplication Levels RNAseq samtools rmdup PCR Duplication $ /Users/nor/analtools/samtools/samtools /samtools faidx /Users/nor/silk/reference/silkcds.fa $ /Users/nor/analtools/samtools/samtools /samtools view -b -o bowmap_silkcds_s_3_index6.sam.bam -t /Users/nor/silk/reference/silkcds.fa.fai bowmap_silkcds_s_3_index6.sam $ /Users/nor/analtools/samtools/samtools /samtools sort bowmap_silkcds_s_3_index6.sam.bam bowmap_silkcds_s_3_index6.sam.sorted $ /Users/nor/analtools/samtools/samtools /samtools rmdup -s bowmap_silkcds_s_3_index6.sam.sorted.bam bowmap_silkcds_s_3_index6.uniq.bam $ /Users/nor/analtools/samtools/samtools /samtools view bowmap_silkcds_s_3_index6.uniq.bam grep HWUSI awk '{print $3}' uniq -c awk '{print $2"\t"$1}' > count_bowmap_silkcds_s_3_index6.uniq.txt SNP sort bam 13349, 12867, 8852, 12734, 10790, 6436, 11927, 10808, 9335, 7642, 11210, 12074, 9257, cdna cdna De novo 52

54 cdna rmdup RNA-seq PCR duplication RNA cdna PCR duplication 53

55 3 GB GB 100GB ( FASTQ ) HDD SSD 1. OS 2. 4G 3. FAT32 NTS- F Mac OS FAT32 windows PC HDD USB FAT32 OS windows PC mac 4GB GB NTSF windows PC mac snow leopard Linux windows PC windows PC mac NTFS 54

56 NTFS_Enabler.txt.zip NTSF Mac OS mac B Time machine HDD UI split bcl bcl FASTQ 4GB 4GB FAT32 FAT32 FAT32 55

57 56

58 チャプタ 4 解析例 De novo アセンブリ シーケンスに失敗したphiXの評価

59 1 De novo transcriptome velvet n0rr:~ nor$ cd /Users/nor/analtools/ n0rr:analtools nor$ mkdir velvet 1. velvet mrna-seq velvet velvet_ tgz /Users/nor/analtools/velvet velvet_ tgz /users/nor/downloads velvet velvet_ tgz n0rr:analtools nor$ cd velvet n0rr:velvet nor$ ls velvet_ tgz n0rr:velvet nor$ tar zxvf velvet_ tgz velvet_ n0rr:velvet nor$ ls velvet_ velvet_ tgz 58

60 velvet_ n0rr:velvet nor$ cd velvet_ n0rr:velvet_ nor$ make MAXKMERLENGTH=63 MAXKMER- LENGTH=63 kmer n0rr:velvet_ nor$ /Users/nor/analtools/velvet/velvet_1.2.03/velveth velveth oases velvet oases /Users/nor/analtools oases oases_ tgz oases n0rr:velvet_ nor$ cd.. n0rr:velvet nor$ cd.. n0rr:analtools nor$ mkdir oases n0rr:analtools nor$ cd oases/ n0rr:oases nor$ mv /Users/nor/Downloads/oases_ tgz./oases_ tgz Finder n0rr:oases nor$ tar zxvf oases_ tgz n0rr:oases nor$ cd oases_ oases_ n0rr:oases_ nor$ make 'VELVET_DIR=/Users/nor/analtools/velvet/velvet_1.2.03/' 'MAXKMERLENGTH=63' velvet kmer n0rr: run_sample_lane3 nor$ /Users/nor/analtools/velvet/velvet_1.2.03/velveth./mac_velvet fastq s_3_1_sequence.txt n0rr: run_sample_lane3 nor$ cd mac_velvet63/ 59

61 n0rr:mac_velvet63 nor$ /Users/nor/analtools/velvet/velvet_1.2.03/velvetg./ -read_trkg yes -scaffolding no oases oases mrna-seq transcripts.fa n0rr:mac_velvet63 nor$ /Users/nor/analtools/oases/oases_0.2.05/oases./ nor$ more contigs.fa sed -e tr -d "\n" tr ">" "\n" awk -F '{print length($2)}' head -n4 head -n 4 nor$ head -n 4 contigs.fa contigs.fa transcripts.fa cdna n cdna nor$ more contigs.fa grep ">" wc -l > \n 1176 length_ kmer awk length contig length_63 nor$ grep length_62_ contigs.fa 60

62 62 63 contig CTGCAGCAGTTAAACCAAAAGCCTCGGCAAAAGCTCCAGGA AAA" wc nor$ more contigs.fa sed -e tr -d "\n" tr ">" "\n" grep _length_63_ awk -F '{print length($2)}' head -n4 nor$ more contigs.fa sed -e tr -d "\n" tr ">" "\n" grep _length_63_ awk -F '{print ">"$1"\n"$2}' head -n4 R nor$ more contigs.fa sed -e '/>/s/$/@/g' tr -d "\n" tr ">" "\n" awk -F "@" '{print length($2)}' tr "\n" "," > length_contigs.txt echo nor$ more transcripts.fa sed -e '/>/s/$/@/g' tr -d "\n" tr ">" "\n" awk -F "@" '{print length($2)}' tr "\n" "," > length_transcripts.txt nor$ more contigs.fa sed -e '/>/s/$/@/g' tr -d "\n" tr ">" "\n" grep _length_63_ awk -F "@" '{print ">"$1"\n"$2}' head -n4 nor$ echo "CTGCAGCAGTTAAACCAAAAGCCTCGGCAAAAGCTCCAGG AAAAGGTGTTAAGAAACCAACAGCAAAGGTGCTGCGAAAC 0,1176,1372,... lcon<c 0 61

63 R, ) lcon<-c(1176,1372,1125,2877,205,...,125,125) summary(lcon) R 62

64 hist(lcon) hist(log(lcon)) contigs.fa transcripts.fa transcipts.fa length_transcripts.txt MASS library(mass) truehist(log(lcon)) 63

65 2 phix Illumina Genome Analyzer Ⅱx Illumina Genome Analyzer Ⅱx FastQC 3. phix phix phix phix FastQC 4. 64

66 phix bowtie phix (-l 65 -n 2) 92.96% SEQanswers 98.5% phix "For comparison PhiX maps at around 98.5% which suggests that the reduction in human is due to genome complexity and not sequence quality." hlight=phix+sequence fastq fasta %, 5 TAAAAAACGTTCNGGCGGTCGCCCTGGTCGTC TGTCTAAAGGTAAAAAACGTTCTGGCGCTCGCCCTGGTCGTCCGCAGCCG ATTTCTAAAGGTAAAAAACGTGCTGGCGCTCG GAGTGATGTAATGTCTAAAGGTAAAAAACGTTCTGGCGCTCGCCCTGGTC ATTTCTACTCTTTCTNAATCCCCAATGCTTTG GGCTTGTGGCATTTCTACTCTTTCTCAATCCCCAATGCTTGGCTTCCATA TCTTGGACGNTTTTTTATGGTTCGTTCTTATT TGCCTCCAAATCTTGGAGGCTTTTTTATGGTTCGTTCTTATTACCCTTCT AATGTCACGNTGATTATTTTGACTTTGAGCCT TACCCTTCTGAATGTCACGCTGATTATTTTGACTTTGAGCGTATCGAGGC phix 0%(0 65

67 FastQC phix 0% poly-a # # FastQC 66