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Collection and Sequencing

Specimen Collection Process

TCRB specimens were collected at clinical sites throughout Texas. Clinical pathologists performed gross inspections of specimens to verify the presence of tumor, then created 50-100 mg aliquots for various uses in the TCRB specimen processing work flow. If the specimen met TCRB requirements for renewable derivative creation, one aliquot was immediately sent for cell line or xenograft creation. Another aliquot was sent for pathological quality control (QC) where the percent tumor cellularity was established and a pathological diagnosis confirmed. The remaining aliquots were stored in regional TCRB biobank facilities for distribution to public researchers.

QC aliquots that passed the pathology quality control checks were shipped to the Nucleic Acids Lab at the Human Genome Sequencing Center for DNA and RNA extraction. The DNA quality and RIN (RNA Integrity) scores were entered into the Acquire database and evaluated by the collection site for process improvement reviews. DNA and RNA meeting the quality criteria could then enter the sequencing queue. All derivatives of the original specimen were stored for future research.

For more information on the TCRB Standard Operating Procedures, please click on the Resources link and view details under “TCRB Forms”.

TCRB specimen workflow

Sequencing Process Description

Specimens were sequenced on Illumina HiSeq machines, with 100 base-pair paired-end reads, to a depth of approximately 200x over the exonic regions. The exome capture design was VCRome 2.1 (see HGSC VCRome Design), supplied by NimbleGen. Mapping was to human reference build 19, verson "GRCh37-lite", using BWA (Burrows-Wheeler Aligner) subsequent base-quality recalibration and local realignment around known indel sites was performed by GATK. See the individual file metadata or BAM headers for exceptions, version numbers, and/or extra information.