Whole Exome Sequencing of Murine Tumor Samples

B16F10 (tumour) cells were expanded in culture to 75% confluence. Total splenocytes (germline) were isolated from a male C57BL/6 colony founder. Genomic DNA was isolated using DNeasy Blood & Tissue kit (Qiagen, cat#: 6950). Whole exome sequencing (WES) libraries were prepared using SureSelectXT Mouse All Exon kits (Agilent, cat# G7550A). Paired-end 100 bp sequencing was performed using HiSeq2500 reagent kit v3 (Illumina, CA) targeted sequencing depths of 300x and 150x for tumour and germline samples, respectively. Sequencing reads were mapped to GRCm38.p6/mm10 using BWA-MEM^{69 (link)}. Duplicate read marking and base quality score recalibration were performed using GATK/Picard^{70 (link)}. Somatic variant calling was performed for target regions using MuTect and Strelka with default filters^{71 (link)}.

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Finnigan J.P., Newman J.H., Patskovsky Y., Patskovska L., Ishizuka A.S., Lynn G.M., Seder R.A., Krogsgaard M, & Bhardwaj N. (2024). Structural basis for self-discrimination by neoantigen-specific TCRs. Nature Communications, 15, 2140.

Publication 2024

DNeasy Blood & Tissue kit SureSelectXT Mouse All Exon kits HiSeq2500 reagent kit v3

Corresponding Organization :

Other organizations : Icahn School of Medicine at Mount Sinai, New York University, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Mount Sinai Hospital

Top 5 similar protocols

Variable analysis

independent variables

Cell line (B16F10 tumour cells)
Isolation of splenocytes (germline) from a male C57BL/6 colony founder

dependent variables

Whole exome sequencing (WES) data for tumour and germline samples

control variables

Genomic DNA isolation using DNeasy Blood & Tissue kit (Qiagen, cat#: 6950)
WES library preparation using SureSelectXT Mouse All Exon kits (Agilent, cat# G7550A)
Paired-end 100 bp sequencing using HiSeq2500 reagent kit v3 (Illumina, CA)
Sequencing depth targets of 300x for tumour and 150x for germline samples
Sequencing read mapping to GRCm38.p6/mm10 using BWA-MEM
Duplicate read marking and base quality score recalibration using GATK/Picard
Somatic variant calling for target regions using MuTect and Strelka with default filters

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