Targeted Sequencing

Data Generation Protocols	Data Analysis Protocols
Data Generation Protocols	Data Analysis Protocols

Data Generation Protocol

The data generation protocols for the Burkitt Lymphoma (BL) project were acquired from the following manuscript.

Grande BM, Gerhard DS, Jiang A, et al. Genome-wide discovery of somatic coding and non-coding mutations in pediatric endemic and sporadic Burkitt lymphoma. Blood. March 2019; 21;133(12):1313-1324. (PMID: 30617194)

Targeted sequencing by custom hybridization capture

Targeted sequencing libraries were constructed from DNA provided by Nationwide Children’s Hospital (Columbus, OH) using a custom hybridization capture protocol. 50 ng from each of 20 or 21 whole genome libraries was pooled prior to custom capture using Agilent SureSelect XT Custom probes (4.8 Mbp) targeting 74,809 human and EBV features (https://cgci-data.nci.nih.gov/PreRelease/BLGSP/targeted_capture_sequencing/DESIGN/). The features included the following: exons of recurrently mutated genes with the exception of known targets of passenger mutations (e.g. TTN, mucin genes); exons of several known diffuse large B-cell lymphoma (DLBCL) genes; exons of previously reported BL genes not found mutated in our data; whole gene bodies for DDX3X (GRCh38 chrX:41332775-41364961) and FBXO11 (chr2:47782639-47907718); whole gene bodies and flanking regions for ID3 (chr1:23557918-23657826) and BCL6 (chr3:187718649-188265924); the recurrently rearranged region surrounding MYC (chr8:127242368-129788153); and non-coding mutation peaks (details below). The pooled libraries were hybridized to the RNA probes at 65°C for 24 hours. Following hybridization, streptavidin-coated magnetic beads (Dynal, MyOne) were used for custom capture. Post-capture material was purified on MinElute columns (Qiagen) followed by post-capture enrichment with 10 cycles of PCR using primers that maintain the library-specific indices. Pooled libraries were sequenced on an Illumina HiSeq 2500 instruments with v4 chemistry generating 125 base paired-end reads.

Experimental Protocols

To request more information or approval regarding the following protocol, please contact BC Cancer at labqa@bcgsc.ca.

Multiplex Agilent Whole Exome or Target Capture

Data Analysis Protocol

The data analysis protocols for the Burkitt Lymphoma (BL) project were acquired from the following manuscript.

Sequencing read alignment

Whole genome sequencing (WGS) and targeted sequencing reads were aligned to the human reference genome (GRCh38) with BWA-MEM (version 0.7.6a; parameters: -M). The human reference genome that was used is a version of GRCh38 without alternate contigs that includes the Epstein–Barr viral genome (GenBank accession AJ507799.2), which can be downloaded at http://www.bcgsc.ca/downloads/genomes/9606/hg38_no_alt/bwa_0.7.6a_ind/genome/. Read duplicate marking was done using sambamba (version 0.5.5). The WGS read alignments for the discovery tumor and matched normal had an average non-redundant depth of 82X (range 55-96) and 41X (range 30-51), respectively. The validation tumor and normal samples were sequenced to a higher average depth, namely 243X (range 158-392). Tumor and matched normal WGS data for 15 cases from the International Cancer Genomic Consortium (ICGC) were obtained through a DACO-approved project using a virtual instance on the Cancer Genome Collaboratory. The ICGC WGS reads were re-aligned using the above parameters, yielding alignments with an average depth of 40X (range 29-62). RNA sequencing (RNA-seq) reads (mean 200M reads; range 100-289M) were pseudo-aligned using Salmon (version 0.8.2; details below). The RNA-seq reads were also aligned to the reference genome indicated above using the JAGuaR pipeline. miRNA sequencing (miRNA-seq) reads (mean 13M reads; range 1.8-35M) were aligned to the same human reference genome with BWA-SW (version 0.5.7).

Sequencing alignment bed files can be found here.

Last updated: June 16, 2020