Similar protocols

Protocol publication

[…] DNAs (four CRPC cases, CRPC1-3 and CRPC5; three CSPC cases, CSPC1-2 and CSPC4) including exonic sequences of 55 cancer genes and 38 introns of 18 genes, where fusion breakpoints have been described using Sure Select Custom DNA Kit (Agilent, Santa Clara, CA, USA) following the manufacturer's recommendations. Since we had very low amounts of input DNA we increased the number of cycles in the enrichment PCR to 20. Six libraries were pooled equimolarily and sequenced on an Illumina MiSeq (Illumina, San Diego, CA, USA)., We generated a mean of 7.78 million reads (range, 3.62-14.96 million), 150 bp paired-end reads on an Illumina MiSeq (Illumina, San Diego, CA, USA). Sequences were aligned using BWA [] and duplicates were marked using picard []. We subsequently performed realigning around known indels and applied the Unified Genotyper SNP-calling software provided by the GATK []., We further annotated resulting SNPs by employing annovar [] and reduced the SNP call set by removing synonymous variants, variants in segmental duplications and variants listed in the 1000 Genome Project [] and Exome sequencing (Project Exome Variant Server, NHLBI Exome Sequencing Project (ESP), Seattle, WA) [] with allele frequency >0.01., We set very stringent criteria to reduce false positives according to previously published values []: a mutation had to be absent from the constitutional DNA sequencing and the sequencing depth for the particular nucleotide position had to be >20-fold. Furthermore, all putative mutations or breakpoint spanning regions were verified by Sanger sequencing., Since plasma DNA is fragmented the read pai […]

Pipeline specifications

Software tools BWA, Picard, GATK, ANNOVAR
Databases Exome Variant Server
Organisms Homo sapiens
Diseases Abnormalities, Multiple, Disease, Neoplasms, Urogenital Neoplasms, Genital Neoplasms, Male, Prostatic Diseases