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Pipeline publication

[…] serous cases and 609 cases of other sub-types from the SCOTROC (SRO), ICON7 (ICN) and OV04 (CAM) ovarian cancer clinical trials., Target sequence enrichment was performed using 48.48 Fluidigm access arrays and 4-primer chemistry for addition of barcode and adapter sequences during the PCR amplification. Target sequence amplicons were 200bp or smaller for complete sequencing using 100 bp pair-end sequencing on the Illumina HiSeq2000 according to the manufacturer's protocol (Illumina Inc, San Diego, CA). A total of 314 amplicons were designed and the average fragment size was 187 base pairs (range 150-200) ()., Sequenced reads were de-multiplexed using standard Illumina software. We used the Burrows-Wheeler Aligner (BWA) [] for sequencing read alignment against the human genome reference sequence (hg19). The Genome Analysis Toolkit (GATK) [] was used for base quality-score recalibration, local insertion/deletion (indel) realignment, and substitution/indel discovery. Variants were only considered for further analysis if they satisfied the set of recommended GATK filters as applicable to our data and as described in the GATK best practices guide. ANNOVAR [] was used to annotate the sequence variation detected. The transcript identifiers used for mutation annotation of the 12 genes are detailed in ., Variant alternate allele frequency was defined as the fraction of alternate allele bases compared to the total number of bases at the variant locus. We applied the following thresholds for variants calling: the minimum coverage is 15, a variant will be called if (1) coverage ≥500 and alternate allele frequency ≥10 percent, or (2) 250 ≤coverage <500 and alternate allele frequency ≥15 percent, or (3) 30 ≤coverage <250 and alternate allele frequency ≥20 percent, or (4) 15 ≤coverage <30 and alternate allele frequency ≥30 percent. The […]

Pipeline specifications

Software tools BWA, GATK, ANNOVAR