SNP Discovery

BCM-HGSC Mutation Discovery Pipeline

Single Nucleotide Mutations

Sequencing reads are compared with their respective amplicon reference sequences using a modification of SNPDetector, which employs a relaxed Het peak ratio threshold to compensate for possible heterogeneity of the tumor tissue sample. We use Polyphred 6.0b as a backup discovery method and capture any high-scoring variation missed (very rarely) by SNPDetector. Special mention should be made of our collaboration with Dr. Jinghui Zhang in the laboratory of Dr Ken Beautow, on the development and calibration of SNPdetector. This software has consistently outperformed other routines for the direct discovery of heterozygotes.

Putative polymorphisms accumulated from this analysis are annotated with the following information:

  1. Chromosome and global coordinates

  2. Coincidence with known variation (dbSNP current build, as well as local databases of newly identified variants)

  3. Functional information:

    1. gene compartment (intron, exon, splice junction)

    2. non-synonymous amino acid change if any

    3. position of non-synonymous amino acid in protein

    4. BLOSSUM62 score of variant amino acid compared to reference

Novel SNPs with recognizable functional potential (e.g., non-synonymous SNP or splice junctional variants) are further evaluated. First, they are visually inspected at the trace level and those that are not clearly noise are passed on to experimental validation, currently pyro-sequencing. We plan to resequence with Sanger reads the matched normal tissue in patients with mutations passing pyro-sequencing validation.

All putative genotypes of each individual at each mutation position, along with annotation and validation status will be stored in local databases. Reports are formatted for submission to common data repositories according to protocols jointly established.

Structural Variation Discovery

We are in the process of evaluating Polyphred 6.0b and a new module for SNPDetector designed for detecting intra-exonic indels in biallelic resequencing traces. One or both of these will be used for indel discovery and characterization. Genotype frequencies of constitutional variants (i.e., known SNPs) will be tracked since they might reveal commonly deleted genes or gene segments (LOH) through departures fro, Hardy-Weinberg equilibrium.

Quality Control

Sequencing coverage is a critical factor leading to variation discovery. We track coverage using the SNPDetector program rather than by a single base quality measure. Bases are judged to be covered in a given read if SNPDetector is able to make a call at any given position regardless of their Phred quality score (although there is a high correlation between Phred quality score and SNPDetector coverage).

Related Publications

McDonough CW, Magvanjav O, Sá ACC, Rouby NMEl, Dave C, Deitchman AN, et al.. Genetic Variants Influencing Plasma Renin Activity in Hypertensive Patients From the PEAR Study (Pharmacogenomic Evaluation of Antihypertensive Responses). Circ Genom Precis Med. 2018 ;11(4):e001854.

Guo L, Akahori H, Harari E, Smith SL, Polavarapu R, Karmali V, et al. CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis. J Clin Invest. 2018 ;128(3):1106-1124.

Brammer DW, Gillespie PJ, Tian M, Young D, Raveendran M, Williams LE, et al. MLH1-rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques. Proc Natl Acad Sci U S A. 2018 ;115(11):2806-2811.

Tanner J-A, Zhu AZ, Claw KG, Prasad B, Korchina V, Hu J, et al. Novel CYP2A6 diplotypes identified through next-generation sequencing are associated with in-vitro and in-vivo nicotine metabolism. Pharmacogenet Genomics. 2018 ;28(1):7-16.

Zhang Y, Guallar E, Ashar FN, Longchamps RJ, Castellani CA, Lane J, et al. Association between mitochondrial DNA copy number and sudden cardiac death: findings from the Atherosclerosis Risk in Communities study (ARIC). Eur Heart J. 2017 ;38(46):3443-3448.

Liu D, Ho M-F, Schaid DJ, Scherer SE, Kalari K, Liu M, et al. Breast cancer chemoprevention pharmacogenomics: Deep sequencing and functional genomics of the ZNF423 and CTSO genes. NPJ Breast Cancer. 2017 ;3:30.

Howson JMM, Zhao W, Barnes DR, Ho W-K, Young R, Paul DS, et al. Fifteen new risk loci for coronary artery disease highlight arterial-wall-specific mechanisms. Nat Genet. 2017 ;49(7):1113-1119.

Rustagi N, Zhou A, W Watkins S, Gedvilaite E, Wang S, Ramesh N, et al. Extremely low-coverage whole genome sequencing in South Asians captures population genomics information. BMC Genomics. 2017 ;18(1):396.