二代测序数据分析简介.ppt

二代测序数据分析简介 童春发 2013.12.23 主要内容 重测序的原理及流程 数据结构与质量评估 SRA数据库及数据获取 Bowtie2、BWA和SAMtools软件使用 重测序的原理及流程 数据结构与质量评估 Fastq格式 FastQC FASTQ format A FASTQ file containing a single sequence might look like this Illumina sequence identifiers With Casava 1.8 the format of the @ line has changed Quality A quality value Q is an integer mapping of p (i.e., the probability that the corresponding base call is incorrect). Phred quality score: The Solexa pipeline (i.e., the software delivered with the Illumina Genome Analyzer) earlier used Quality Encoding Sanger format can encode a Phred quality score from 0 to 93 using ASCII 33 to 126 Illuminas newest version (1.8) of their pipeline CASAVA will directly produce fastq in Sanger format Solexa/Illumina 1.0 format can encode a Solexa/Illumina quality score from -5 to 62 using ASCII 59 to 126 Starting with Illumina 1.3 and before Illumina 1.8, the format encoded a Phred quality score from 0 to 62 using ASCII 64 to 126 Starting in Illumina 1.5 and before Illumina 1.8, the Phred scores 0 to 2 have a slightly different meaning American Standard Code for Information Interchange (ASCII) FastQC http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Double click “run_fastqc.bat” to run FastQC The analysis results for 11 modules Green tick for normal Orange triangle for slightly abnormal Red cross for very unusual Basic Statistics Per Base Sequence Quality Per Sequence Quality Scores Per Base Sequence Content Per Base GC Content Per Sequence GC Content Per Base N Content Sequence Length Distribution Duplicate Sequences Overrepresented Sequences Overrepresented Kmers Saving a Report SRA数据库及数据获取 SRA数据库及数据获取 SRA数据库及数据获取 SRA数据库及数据获取 查看和下载SRR576183 Fastq-dum将SRA文件转化成FASTQ格式 fastq-dump --split-files -DQ “+” ./SRR576183.sra fastq-dump --split-files -DQ “+” --gzip ./SRR576183.sra 直接下载FASTQ格式数据 ftp://ftp.era.ebi.ac.uk/vol1/fastq/SRR576/SRR576183 将Reads比对到参考序列 BWA Bowtie2 Soap Samtools BWA / /lh3/bwa wget /projects/bio-bwa/files/