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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454Illumina 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,Bridge PC
2、R,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 SYNTHESIS OVE
3、RVIEW,CYCLIC REVERSIBLE TERMINATION,All four labeled reversible terminators are added per cycleRemove unincorporated bases and detect signalRemove the terminating group and the fluorescent dye,Trends in Genet 24:133(2008),Terminating group,Fluorophore cleavage,Nat Rev Genet 11:31(2010),BASE CALLING,
4、FLOWCELL LAYOUT ON GAII,A flow cell contains 8 lanes,Lane 1,Lane 2,Lane 8,.,Column 1Column 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,Firecrest,Bustard,Seq
5、uence 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,MATE-PAIR 建库和测序,Molecula
6、r Ecology Resources(2011),TEMPLATE PREPARATION-EMULSION PCR,Trends in Genet 24:133(2008),PYROSEQUENCING,Single dNTP type flows per cycleInorganic pyrophosphate(PPi)drives visible light through a series of reactionsRemove unincorporated nucleotide,Trends in Genet 24:133(2008),BASE CALLING,Homopolymer
7、 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 manufacturingSequencing 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，或者和第一行一样 第四行是第二行序列质量数据的编码，
8、长度需和第二行一样,HWI-ST507:211:C18E6ACXX:2:1101:1688:1992 1:N:0:GAGTGGCGACAATTTTTTTTGATATTAATAAAGATAGAACTTTCTTCCTATGAGTTTTCTCTC+CCCFFDFFHHHHGJJGHIIJGIIJJJJIIJJHJJJJJIJJIIIGIIIJGGIHJDIJIGAHEHFFGHGHE,Example:,ILLUMINA SEQUENCE IDENTIFIERS,HWI-EAS364_0004:4:1:995:9044#0/1,Casava 1.8以前的序列标识,ILLUMINA SEQUENCE I
9、DENTIFIERS,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.JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ.LLLLLLLLLLLLLL
10、LLLLLLLLLLLLLLLLLLLLLLLLLLLL.!#$%?ABCDEFGHIJKLMNOPQRSTUVWXYZ_abcdefghijklmnopqr|33 59 64 73 1040.26.31.40-5.0.9.40 0.9.40 3.9.40 0.26.31.41 S-Sanger Phred+33,raw reads typically(0,40)X-Solexa Solexa+64,raw reads typically(-5,40)I-Illumina 1.3+Phred+64,raw reads typically(0,40)J-Illumina 1.5+Phred+64
11、,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 length=181 xy=1540_3788 region=1 run=R_2012_08_01_00_39_39ACGTGTTCTGAGC
12、CATATTGCGGTACTGGAAGGTGCGCCTGCACTGTCTGAGCACTGGTCACTGCTCGATACCAATGAAGCCTTATTTGATGAGGCGCGCACCACGCAGGCGGCGACTATTATCTTCTCGTTTGATCCAGAATAACCAAATCGAAAACGCTGGCAAGGCACACAGGGGATAHSAPGDX01D1KDA length=181 xy=1540_3788 region=1 run=R_2012_08_01_00_39_3940 40 40 40 40 40 40 39 37 38 36 34 24 23 19 19 19 24 20 19
13、 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 30 31 30 28 28 30 31 31 32 32 31 31 30 30 30 31 27 24 24 22 20 20 20 22 262
14、6 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 by sequencer.Reads,sequenced fragments.Read length and quality.Coverage fol
15、d,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 biologySIBS,Chinese Academy 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 biologySIBS,Chinese Academy of S
16、ciences,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 Adaptor/primer polyA|T region Overall low-complexity sequence(Dust)Contamination/unwanted sequences
17、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reads5接头污染的reads没 有插入判断的reads,
