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Protocol publication

[…] the 10x objective. Cell confluence was calculated with the IncuCyte ZOOMTM software (version 2015A)., A customized NGS panel targeting 222 cancer related genes was used to sequence DNA. DNA Probes for capturing exon regions in these genes were manufactured by Roche NimbleGen. SeqCap EZ Library SR User’s Guide (Roche, Pleasanton, CA) was followed for library preparation and capture of targeted sequences. Paired-end sequencing of 2 × 150 bp was performed on a MiSeq platform (Illumina, San Diego, USA). Twelve individual libraries were multiplexed for a MiSeq flow cell. The mean sequencing depth of coverage was 135x., Paired-end reads were aligned to the GRCh37 version of the human genome using Burrows-Wheeler Aligner v0.7 to generate BAM files. After sorting the BAM files using samtools, PCR duplicates marked using Picard and realignment around putative gaps was performed using the Genome Analysis Toolkit (GATK) v3.2-2. Variant calling was performed with the GATK Haplotype caller. ANNOVAR ( was used for annotating variants and for retrieving information on variants in the population-based studies such as the 1000 Genomes Project (, NHLBI-ESP 6500 exomes or ExAC (, and clinical databases such as the Human Gene Mutation Database (HGMD) and ClinVar. Pathogenicity of variants is defined based on American College of Medical Genetics and Genomics (ACMG) criteria. Specifically, pathogenic and likely pathogenic mutations are defined as (1) all protein truncating mutations unless their allele frequency is 5% or higher in any racial group in population databases or is reported as benign or likel […]

Pipeline specifications

Software tools BWA, SAMtools, Picard, GATK, ANNOVAR
Databases HGMD
Organisms Homo sapiens
Diseases Neoplasms, Urogenital Neoplasms, Genital Neoplasms, Male, Prostatic Diseases
Chemicals Ribonucleotides, Heterocyclic Compounds, 2-Ring, Purine Nucleotides, Monosaccharides, Selenium, Chalcogens, Heterocyclic Compounds, 2-Ring, Pyrans