唐主任-miseq应用初步经验

1、MiSeq Implementation in CGSexperience sharingTong Ming For16 August 2014OutlinesData Producing Selection of targeted panel Preparation of libraries Sequencing inside the MiSeqData Processing (WL Lab) Computing analysisData Analysis bioinformaticsFuture endeavorOutlinesData Producing Selection of tar

2、geted panel Preparation of libraries Sequencing inside the MiSeqData Processing (WL / HKU) Computing analysisData Analysis bioinformaticsFuture endeavor.fastq.els.bam.vcf.fastq.els.bam.vcfSummary for reads distributionSampleIDAverage Depth%covered16625_S7_L00148.362794460.9598218070_S3_L00145.054389

3、770.95585118443_S8_L00150.216824080.95729318531_S1_L00151.526956010.95599217445_S5_L00152.512954320.95780616624_S6_L00151.336469320.95838715786_S10_L00150.109462030.95677915854_S11_L00149.330004410.9566217239_S9_L00149.646997330.95618317740_S4_L00149.084928890.95634518313_S2_L00148.56157770.95672912

4、829_S12_L00148.77684270.95726One SNP reported by Miseq result (FBN1:NM_000138:exon66:c.C8234G:p.S2745C)POSREFALTGenotypeADchr1548703569GCHET54,7chr1548703569GTHET54,5All reads mapped in the position, including duplicated reads and reads with bad MQ.fastq.exl.bam.vcfIf we removed those duplication an

5、d reads with low MQ, the result should be ref/refonly 4 reads with G covered in the site, it is more likely to be a hom instead of het.pMiseq automatic pipeline did not remove duplication and used bad MQ reads for genotyping.pBy contrast, if we remove duplicates and used reads with MQ50), only 8 for

6、 G left, but 4 of them are duplications in this example.Not sufficient coverage!pC, T was moved due to low MQ.OutlinesData Producing Selection of targeted panel Preparation of libraries Sequencing inside the MiSeqData Processing (HKU) Computing analysisData Analysis bioinformaticsFuture endeavorPath

7、ogenic by ClinVarBenign by ClinVarc.6380-12_c.6380-13insTData Producing Selection of targeted panel Preparation of libraries Sequencing inside the MiSeqData Processing (WL Lab) Computing analysisData Analysis bioinformaticsBased on fastq file, total produced 1.2 Gb data from 12 samples !Future Endeavor Data Producing Selection of targeted panel Preparation of libraries Sequencing inside the MiSeqData Processing (CGS ?) Computing analysis Data Analysis bioinformaticsFuture EndeavorShort ter