1、二代測序2012.9.201資源數(shù)據(jù)源：http:/http:/數(shù)據(jù)下載方式AsperaFTP數(shù)據(jù)分析交流網(wǎng)站2處理工具匯總http:/ bowtie， bowite2， MUMmer，SOAP，MAQ，bioscopeSNP分析工具： samtools，gatk， SOAPsnp轉(zhuǎn)錄組從頭組裝：Trinity，Velvet Oasis數(shù)據(jù)質(zhì)量分析工具：Fastx-toolkit工具包二代數(shù)據(jù)可視化工具：IGV，Savant，samtools，Gbrowser3二代數(shù)據(jù)結(jié)果輸出格式SAM，BAMhttp:/ example: a line in SAM BWA:HWUSI-EAS172:628

2、C8:4:1:1138:2718 16 chr20 46264803 37 76M * 0 0 ACCCAAGTAAAGTAAGCAATCAGGATTCCAAGAGTCCTCTGGGCGTTTATTGCGACCAAAATCCAGTGGGGAGTTC #?:?=:?ABC?5:8(6:*0:42C-=?C:D:D:(:)1=:DDDBD=:?DD-DDD;B;66= XT:A:U NM:i:3 X0:i:1 X1:i:0 XM:i:3 XO:i:0 XG:i:0 MD:Z:1A42T24A64SAM主體部分1.QNAME，read 名字2.FLAG， bitwise flag，標識read ma

3、p到染色體上的情況3.RNAME，染色體名字4.START，map到染色體上的第一個位置5.MAPPING QUALITY，mapping的質(zhì)量6.CIGAR，比對結(jié)果情況描述（H，S，M）5SAM主體部分7.MRNM，配對read的名字8.MPOS，配對序列的起始位點9.ISIZE，兩個reads間最遠堿基的距離10.SEQQuery：read調(diào)整到與參考基因組同鏈的序列11.reference QUAL，read的質(zhì)量（ASCII-33）6SAM FLAG0X0001 = 1 the read is paired in sequencing0X0002 = 2 the read is ma

4、pped in a proper pair0X0004 = 4 the query sequence itself is unmapped0X0008 = 8 the mate is unmapped0X0010 = 16 strand of the query0X0020 = 32 strand of the mate0X0040 = 64 the read is the first read in a pair0X0080 = 128 the read is the second read in a pair0X0100 = 256 the alignment is not primary

5、0X0200 = 512 QC failure0X0400 = 1024 optical or PCR duplicate7SAM格式附加部分NM Edit distance 編輯距離，與參考基因組的差異堿基數(shù)目MD mismatching positions/bases 錯配的堿基或位置X0 最優(yōu)匹配位置的數(shù)目X1 次優(yōu)匹配位置的數(shù)目XN 參考基因組中模糊堿基的數(shù)目（N）XM 錯配堿基的數(shù)目XO 打開的gap數(shù)目XG 打開的gap中延伸的堿基數(shù)目XT Type：Unique/Repat/N/Mate-sw XA 其他mapping位置報告8二代數(shù)據(jù)分析流程9Sra格式數(shù)據(jù)解壓fastq-du

6、mp option -A / -accession賦予解壓文件新的名字-split-3 分割雙端測序數(shù)據(jù)Order1) fastq-dump -split-3 SRR427121.lite.sra10Read filterFastx-Toolkit1) $ fastx_quality_stats fastx_quality_stats -h -i INFILE -o OUTFILE2) $ fastq_quality_boxplot_graph.shINPUT.TXT -t TITLE -p -o OUTPUT3) $ fastx_trimmer -h -f N -l N -z -v -i I

7、NFILE -o OUTFILE4) $ fastx_nucleotide_distribution_graph.sh-p -i INPUT.TXT -o OUTPUT -t TITLE5) $ fastx_trimmer -h -f N -l N -t N -m MINLEN -z -v -i INFILE -o OUTFILE6) $ fastq_quality_trimmer -h -v -t N -l N -z -i INFILE -o OUTFILEhttp:/fastx_toolkit/galaxy.html11Reads Quality Statsfastx_quality_st

8、ats -i in.fastq -o out.stat12Shortgun reads trimTrimed Sitefastq_quality_boxplot_graph i out.stat o output t title13Fastx-toolkit 實踐Order2) nohup fastx_quality_stats -i SRR427121_1.fastq -o SRR_1.stat -Q33 &Order3.1) fastq_quality_boxplot_graph.sh -i SRR_1.stat -o SRR_1.png D:花SRR_1.pngOrder3.2) fas

9、tx_nucleotide_distribution_graph.sh -i *stat -o SRR_1_nucleotide_distribution D:花SRR_1_nucleotide_distribution.png14Fastax-toolkit結(jié)果分析15Fastx-toolkit結(jié)果分析Trimed Sitefastq_quality_boxplot_graph i out.stat o output t title16Trim fastqfastq_quality_trimmer -t 20 -l 15 -i SRR427121_1.fastq -o ecoli_1.fq

10、-Q33fastq_quality_trimmer -t 20 -l 15 -i SRR427121_2.fastq -o ecoli_2.fq -Q3317Reference genome mapping: BWA1) 建立索引bwa index -p prefix -a algoType -c -p 建立的索引的名字-a 構(gòu)建索引使用的算法，is試用的基因組長度2GB，bwtsw適合的基因組長度10MB-c構(gòu)建color-space 索引，適合solid數(shù)據(jù)比對bwa index -p Ecoli -a is NC_000913.fna2)alnbwa aln -n maxDiff -o

11、maxGapO -e maxGapE -d nDelTail -i nIndelEnd -k maxSeedDiff -l seedLen -t nThrds -cRN -M misMsc -O gapOsc -E gapEsc -q trimQual perl get_consesus_read.pl ecoli_1.fq ecoli_2.fq ecoli_1_trim.fq ecoli_2_trim.fq ecoli_trim.fq& bwa aln -t 10 ./././chromosome/Ecoli ./ecoli_1_trim.fq -f ecoli_1_trim.sai&bwa

12、 aln -t 10 ./././chromosome/Ecoli ./ecoli_2_trim.fq -f ecoli_2_trim.sai&bwa aln -t 10 ./././chromosome/Ecoli ./ecoli_trim.fq -f ecoli_trim.sai18Reference genome mapping: BWA3) samsebwa samse -n maxOcc bwa samse ./././chromosome/Ecoli ecoli_trim.sai ./ecoli_trim.fq -f ecoli_trim.sam4) sampebwa sampe

13、-a maxInsSize -o maxOcc -n maxHitPaired -N maxHitDis -P bwa samse ./././chromosome/Ecoli ecoli_1_trim.sai ./ecoli_1_trim.fq ecoli_2_trim.sai ./ecoli_2_trim.fq -f ecoli_trim_paired.sam19Reference genome mapping：bowtie建立索引bowtie-build options* -f reference input files (fasta)-c reference from command

14、line-C/-color color base (for solid)bowtie-build -f NC_000913.fna ecoli比對bowtie options* -1 -2 | -12 | 20Bowtie 必須文件 參數(shù)bowtie options* -1 -2 | -12 | -1 逗號分隔的文件 seg1-2 逗號分隔的文件 seq2-12 構(gòu)建的以tab鍵分隔的文件-q fastq格式文件-f fasta格式文件r 只有序列的文件,每行一條序列S single end reads-C color base文件比對-Q with f and C-Q1 -Q2 combin

15、ation with f -1 and C-integer-quals -solexa1.3-quals -solexa-quals -phred33-quals -phred64-quals21Alignment 參數(shù)選擇-v 允許的最大mismatch的數(shù)目-l seed 的長度影響速度，l越大，速度越快-n seed 中允許的錯配數(shù)-I 配對序列允許的最小插入長度-X 配對序列允許的最大插入長度-fr 默認533522Output參數(shù)選擇-k 限制為每個read輸出的最大mapping 位置數(shù)目-a 報告全部的mapping 位置-m 不報告mapping位置大于m的read-M 隨機報告mapping位置大于M的mapping結(jié)果-best 確保bowtie匯報最好的mapping(only for single end read)-strata 相當于