1、二代测序的建库与测序原理,何有裕 上海生物信息技术研究中心 上海众信生物技术有限公司 苏州众信生物技术有限公司,内容,样本处理与测序原理简介 罗氏454 Illumina solexa 原始数据质量控制,TruSeq RNA and DNA Sample Preparation,Cluster Generation Overview, 1000-6000 molecules per cluster,OH,Cluster Generation, Template Hybridization,diol,diol,1st cycle denaturation,Cluster Generation
2、, Bridge PCR,Template preparation-bridge RCR,Adaptor ligation,Surface attachment,Bridge amplification,Denaturation,Trends in Genet 24:133(2008),First base incorporated,Cycle 1: Add sequencing reagents,Detect Signal,Cleave Terminator and Dye,Cycle 2-n: Add sequencing reagentsand repeat,Sequencing by
3、Synthesis Overview,Cyclic reversible termination,All four labeled reversible terminators are added per cycle Remove unincorporated bases and detect signal Remove the terminating group and the fluorescent dye,Trends in Genet 24:133(2008),Terminating group,Fluorophore cleavage,Nat Rev Genet 11:31(2010
4、),Base calling,Flowcell layout on GAII,A flow cell contains 8 lanes,Lane 1,Lane 2,Lane 8,. . .,Column 1 Column 2,Each lane contains 2 columns,Each column contains 60 tiles,Each tile is imaged 4 times per cycle,Primary Data Analysis By Firecrest and Bustard in RTA/OLB,tiff image file,Intensity file,F
5、irecrest,Bustard,Sequence file,OH,diol,diol,OH,Cluster Generation, Sequencing Primer Hybridization（Single测序方式处理步骤）,Sequence multiple samples in the same lanes,DNA insert,Read 1,Index Read,Read 2,DNA insert,Index,Index SP,Rd2 SP,Rd1 SP,Multiplexing multiple samples in the same lanes,Pair-end 测序优势,Mat
6、e-pair 建库和测序,Molecular Ecology Resources (2011),Template preparation- emulsion PCR,Trends in Genet 24:133(2008),Pyrosequencing,Single dNTP type flows per cycle Inorganic pyrophosphate (PPi) drives visible light through a series of reactions Remove unincorporated nucleotide,Trends in Genet 24:133(200
7、8),Base calling,Homopolymer error,GV6330,20,灵活的多样本标签技术,454、solexa测序模式,Detect H+ released as a voltage changefast Common microchip design standardslow-cost manufacturing Sequencing volume is increasing,Semiconductor sequencing,Fasta序列格式,Fastq 文件用4行记录一条序列,第一行以字符开头，跟在后面的是序列标识和描述第二行是序列字符第三行以+字符开头，后面可以为空
8、，或者和第一行一样第四行是第二行序列质量数据的编码，长度需和第二行一样,HWI-ST507:211:C18E6ACXX:2:1101:1688:1992 1:N:0:GAGTGG CGACAATTTTTTTTGATATTAATAAAGATAGAACTTTCTTCCTATGAGTTTTCTCTC + CCCFFDFFHHHHGJJGHIIJGIIJJJJIIJJHJJJJJIJJIIIGIIIJGGIHJDIJIGAHEHFFGHGHE,Example:,Illumina sequence identifiers,HWI-EAS364_0004:4:1:995:9044#0/1,Casava 1
9、.8以前的序列标识,Illumina sequence identifiers,HWI-ST507:211:C18E6ACXX:2:1101:1688:1992 1:N:0:GAGTGG,Casava 1.8的序列标识,序列质量,附：Solexa 1.3以前的quality计算公式是:,SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS. XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX .IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII .JJJJJJJJJJJJJJJJJJJJJJ
10、JJJJJJJJJJJJJJJJJ LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL !“#$%?ABCDEFGHIJKLMNOPQRSTUVWXYZ_abcdefghijklmnopqr | | | | | 33 59 64 73 104 026.31.40 -509.40 09.40 3.9.40 026.3141S - Sanger Phred+33, raw reads typically (0, 40)X - Solexa Solexa+64, raw reads typically (-5, 40)I - Illumina 1.3+ Phred+
11、64, raw reads typically (0, 40)J - Illumina 1.5+ Phred+64, raw reads typically (3, 40)with 0=unused, 1=unused, 2=Read Segment Quality Control Indicator (bold) (Note: See discussion above).L - Illumina 1.8+ Phred+33, raw reads typically (0, 41),Q值对应ASCII码,454原始数据图片、sff格式、fasta格式（qual）,HSAPGDX01D1KDA
12、length=181 xy=1540_3788 region=1 run=R_2012_08_01_00_39_39 ACGTGTTCTGAGCCATATTGCGGTACTGGAAGGTGCGCCTGCACTGTCTGAGCACTGGTCACTGCTCGATACCAATGAAGCCTTATTTGATGAGGCGCGCACCACGCAGGCGGCGACTATTATCTTCTCGTTTGATCCAGAATAACCAAATCGAAAACGCTGGCAAGGCACACAGGGGATA HSAPGDX01D1KDA length=181 xy=1540_3788 region=1 run=R_2012_
13、08_01_00_39_39 40 40 40 40 40 40 40 39 37 38 36 34 24 23 19 19 19 24 20 19 18 18 26 26 18 18 19 18 20 20 20 25 25 26 19 20 20 22 22 22 25 28 26 24 22 22 22 25 24 28 28 28 29 29 28 30 30 30 26 2626 27 27 27 31 31 30 28 28 28 30 30 30 30 26 21 21 20 20 26 27 28 24 25 20 20 20 20 19 19 19 27 28 28 30 3
14、0 31 30 28 28 30 31 31 32 32 31 31 30 30 30 31 27 24 24 22 20 20 20 22 2626 22 22 23 16 16 16 19 22 16 13 13 13 16 22 23 23 23 26 26 24 24 26 13 13 11 11 12 12 19 22 18 18 11 11 13 13 18 24 24 24 24 26 26 26 27 29 29 31 33 32 31 31 27 27 27 29 29 28 2622,454原始数据长度分布（质控后一样）,Yield, data size produced
15、by sequencer. Reads, sequenced fragments. Read length and quality. Coverage fold, number of times a nucleotide is represented. Depth, the average coverage fold. Coverage rate, ratio of the region sequenced to the whole genome. Homopolymer, e.g. AAAAA,Key lab of systems biology SIBS, Chinese Academy
16、of Sciences,一些测序中提到的基本概念,通常深度测序数据处理流程,Key lab of systems biology SIBS, Chinese Academy of Sciences,序列质量评估, FastQC: A quality control tool for high throughput sequence data Java http:/www.bioinformatics.bbsrc.ac.uk/projects/fastqc/ Function:,QC pipeline,原始数据的质控过滤,Sequence level Short sequences Adapto
17、r/primer polyA | T region Overall low-complexity sequence (Dust) Contamination/unwanted sequences Ns (low quality ends) Quality level Low quality base or region 目标：所有保留的都是高质量的，真正参与生物信息分析的数据。,Clean reads,去掉含有接头序列的reads； 当单端测序read中含有的N的含量超过该条read长度比例的10% 时,去除此对paired reads； 当单端测序read中含有的低质量(低于5)碱基数超过该条read长度比例的50% 时，需要去除此对paired reads。,Reads中不合格的碱 基判断标准： reads中出现N， 记个数 reads中碱基质量分数低于20分， 记个数 去除的reads条件： 质 量不合格的碱基占reads长度的10%以 上（即10bp） 没 有3接 头的reads 5接头污染的reads 没 有插入判断的reads,
