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Protocol publication

[…] dow of the mean. CREST (Clipping Reveals Structure) was used on WGS data to identify structural variations ., RNAseq data was aligned against human reference genome (build 36) with TopHat 1.2; RNAseq reads were only aligned against the autosomes and sex chromosomes. Mitochondrial DNA and annotations were removed from the genome and annotation references prior to alignment. Cuffdiff was used to identify differentially expressed genes and isoforms. Differential analysis was performed on FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) expression values calculated for gene and isoform. P-values were corrected for multiple testing using the Benjamini and Hochberg method. ChimeraScan was used for fusion transcript detection., Integrative analysis of whole genome and transcriptomic data was performed using the Functional Ontology Enrichment Tool in MetaCore from GeneGo, Inc. (v6.8; Thomson Reuters Business, Philadelphia, PA). Pathway analysis specific to pancreatic cancer was performed using the MetaMiner (Oncology) Pancreatic Cancer Disease Module add-on. P-values associated with each analysis are calculated in MetaCore using a hypergeometric distribution., Our study was performed on a set of fresh pancreatic tumor specimens and whole blood samples from three patients diagnosed with PAC. Clinical information is listed in . For each patient, we sequenced both tumor DNA, as well as germline DNA isolated from whole blood in order to identify somatic changes in the tumors. Read alignment was performed with BWA using build 36 of the human reference genome. WGS metrics and summary statistics for each of the three patients are shown in . Using sequencing by synthesis technology and 100 bp paired end chemistry, we generated nearly 8 billion total reads from WGS for average mapped coverages ranging from 31× to 54×. SNP calling was performed using two separate callers to reduce the false negative rate. To evaluate the overall quality of variant data, germline SNPs were called and the transition to transversion and dbSNP (Single Nucleotide Polymorphism Database) 129 concordance ratios were calculated. For all three patients, the transition/transversion ratios were in the range of 2.01 to 2.24, and t […]

Pipeline specifications

Software tools chimerascan, MetaCore, BWA