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1、 蘇州貝斯派生物科技有限公司 Base pair Genome re-sequencing data analysis:from reads to variants Chao Li,PhD SmartQuerier Biotechnology 蘇州貝斯派生物科技有限公司 Base pair Types of Genetic Variation Single Nucleotide Aberrations Single Nucleotide Polymorphisms(SNPs)-mutations shared amongst a population Single Nucleotide Var

2、iations(SNVs)-private mutations Short Insertions or Deletions(indels)Copy Number Variations(CNVs)Structural Variations(SVs)Large insertions and deletions Inversion Translocation 蘇州貝斯派生物科技有限公司 Base pair Re-sequencing strategy Whole genome sequencing vs Whole exome sequencing Cost Coverage Depth Varia

3、nts type 蘇州貝斯派生物科技有限公司 Base pair Analysis Outline Quality control(QC)Mapping SNP and small INDEL calling Structural variation calling Copy number variation calling Variants function annotation 蘇州貝斯派生物科技有限公司 Base pair Analysis Outline Quality control(QC)Mapping SNP and small INDEL calling Structural

4、variation calling Copy number variation calling Variants function annotation 蘇州貝斯派生物科技有限公司 Base pair FASTQ format Sample_GGCTAC_L004_R1_001.fastq.gz Sample_GGCTAC_L004_R2_001.fastq.gz header line:SEQUENCE_ID sequence line line beginning with+quality score line Illumina raw reads are stored in fastq

5、format Paired-end reads are stored in matched files 4 lines per read 蘇州貝斯派生物科技有限公司 Base pair Illumina identifier HWUSI-EAS100R the unique instrument name 6 flowcell lane 73 tile number within the flowcell lane 941 x-coordinate of the cluster within the tile 1973 y-coordinate of the cluster within th

6、e tile#0 index number for a multiplexed sample(0 for no indexing)/1 the member of a pair,/1 or/2(paired-end or mate-pair reads only)HWUSI-EAS100R:6:73:941:1973#0/1 蘇州貝斯派生物科技有限公司 Base pair Illumina identifier(1.4+)EAS139 the unique instrument name 136 the run id FC706VJ the flowcell id 2 flowcell lan

7、e 2104 tile number within the flowcell lane 15343 x-coordinate of the cluster within the tile 197393 y-coordinate of the cluster within the tile 1 the member of a pair,1 or 2(paired-end or mate-pair reads only)Y Y if the read is filtered,N otherwise 18 0 when none of the control bits are on,otherwis

8、e it is an even number ATCACG index sequence EAS139:136:FC706VJ:2:2104:15343:197393 1:Y:18:ATCACG http:/en.wikipedia.org/wiki/FASTQ_format 蘇州貝斯派生物科技有限公司 Base pair Multiplex A unique barcode is attached to sequence fragments from the same sample.Different samples can be mixed for sequencing to improv

9、e throughput.The sequences could be de-multiplexed and assigned to different samples in Illumina data analysis software.CACTC CGACA TGCGT TGCGT 蘇州貝斯派生物科技有限公司 Base pair Phred score Phred score:if p is the error probability for a base ASCII code for Phred score:Qphred+33(Sanger score)Sanger score is d

10、efault for most software.Phred score Error probability ASCII code 0.9 1.0.13!”#$%&()*10.19 0.1.0.013+,-./01234 20.29 0.01.0.013 56789:;30.39 0.001.0.0013?ABCDEFGH 40 0.0001 I pQphred10log10 蘇州貝斯派生物科技有限公司 Base pair 蘇州貝斯派生物科技有限公司 Base pair Score code space Two main code space:+33 and+64.The space can

11、be distinguished from low-quality tail of data.Latest version of Illumina score(1.8)is nearly the same as Sanger score.http:/en.wikipedia.org/wiki/FASTQ_format 蘇州貝斯派生物科技有限公司 Base pair FastQC Quick impression of whether the data has problems Import of data from FastQ,BAM,SAM Export of results to an H

12、TML based permanent report Available on Linux,Windows etc Command-line or GUI http:/www.bioinformatics.babraham.ac.uk/projects/fastqc/蘇州貝斯派生物科技有限公司 Base pair Per base sequence quality Base quality is generally decreased from 5 to 3 end.Trim low quality ends if they exist.蘇州貝斯派生物科技有限公司 Base pair Per

13、sequence quality score Sequences with low quality needs to be filtered 蘇州貝斯派生物科技有限公司 Base pair Per base GC content Unstable GC content may indicate overrepresented sequences,including Illumina adapters and primers.蘇州貝斯派生物科技有限公司 Base pair Duplicated sequences 蘇州貝斯派生物科技有限公司 Base pair NGS QC Toolkit Pa

14、rallelization Paired-end data integrity Auto detection of FASTQ quality format Processing compressed files PLoS ONE 2012,7(2):e30619 http:/www.nipgr.res.in/ngsqctoolkit.html 蘇州貝斯派生物科技有限公司 Base pair Comparison before vs.after QC 蘇州貝斯派生物科技有限公司 Base pair Analysis Outline Quality control(QC)Mapping SNP

15、and small INDEL calling Structural variation calling Copy number variation calling Variants function annotation 蘇州貝斯派生物科技有限公司 Base pair Issues in genome mapping Genome is large and repetitive misaligned reads will result in false positive variant calls Accuracy Sequenced genome will differ from the

16、reference due to variations Differences between the individual and reference should be allowed to find the correct mapping position Sensitivity NGS instruments produce huge amounts of data With large data the informatics cost is significant Speed 蘇州貝斯派生物科技有限公司 Base pair Mapping algorithm Nat Biotechnol.2009,27(5):455 蘇州貝斯派生物科技有限公司 Base pair SAM/BAM format SAM(Sequence Alignment Map)format represents the alignment of reads to a reference sequence Simple to read and parse(text,tab-delimited)Flexib