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Protocol publication

[…] A FFPE Tissue Kit (Qiagen)., Targeted sequencing was performed for 71 genes, which are listed in . Sixty-one of the genes were previously screened by whole-exome sequencing, while 6 were the family genes of those whose mutations were identified by the whole-exome sequencing. The other four genes were deemed susceptible to mutations in PTCLs on the basis of the mutational profiles of other lymphoid malignancies., , , , , All the exons of the selected genes were captured by use of a SureSelect Target Enrichment Kit (Agilent, Santa Clara, CA, USA) and then massively sequenced using HiSeq2000 (Illumina, Santa Clara, CA, USA). For each sample, all the sequencing reads were aligned to hg19 using BWA version 0.5.8 with default parameters. After all the duplicated reads and the low-quality reads and bases were removed, the allele frequencies of single-nucleotide variants and indels at each genomic position were calculated by enumerating the relevant reads using SAMtools ( Initially, all the variants showing allele frequencies >0.02 were extracted and annotated using ANNOVAR, for further consideration, if they were found in >6 reads of >10 total reads and appeared in both the positive- and the negative-strand reads. All synonymous variants and known single-nucleotide polymorphisms in public and private databases, including dbSNP131, the 1000 genomes project as of 2012/05/21 and our in-house database, were removed. To exclude germline variants, nonsynonymous variants were excluded when the allele frequencies were from 0.45 to 0.55. Candidate mutations were validated by amplicon-based deep sequencing using Ion PGM (Life Technologies, Carlsbad, CA, USA) and/or Sanger sequencing (see below)., In the cohort of 87 cases, 79 were analyzed for RHOA, TET2, […]

Pipeline specifications

Software tools BWA, SAMtools, htslib, ANNOVAR