Identification of novel SNPs in differentially expressed genes and its association with horn cancer of Bos indicus bullocks by next-generation sequencing.

P G Koringa,C G Joshi,S J Jakhesara,D N Rank

doi:10.1007/s13205-015-0351-0

Abstract

The use of polymorphic markers like SNPs promises to provide comprehensive tool for analysing genome and identifying genomic regions that contribute to cancer phenotype. Horn cancer is the most common cancer among Bos indicus animals. Increased expression of some genes due to polymorphisms increases risk of HC incidence. We successfully amplified 91 SNPs located in 69 genes in 52 samples, each of HC and HN. Equimolar concentration of amplicons from 69 PCR products of each sample was pooled and subjected to sequencing using Ion Torrent PGM. Data obtained were analysed using DNASTAR software package and case control analysis using SAS software. We found SNP present in BPIFA1 gene of B. indicus shows association with event of HC which reflects its potential to be a genetic marker. Bioinformatic analysis to detect structural and functional impact nsSNP of BPIFA1 added another layer of confirmation to our result. We successfully identified SNP associated with HC as well as demonstrated efficient approach for limited number of SNP discovery and validation in targeted genomics regions in large number of samples combining PCR amplification and Ion Torrent PGM sequencing which suits small-scale laboratories with limited budget.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-015-0351-0) contains supplementary material, which is available to authorized users.

Highlights

Recent advances in sequencing technologies give unrivalled opportunities to explore individual genomic landscapes and identify mutations related to cancer, a complex set of diseases characterised by both environmental and genetic contributions
To validate the single-nucleotide polymorphisms (SNPs) identified in our previous study as well as to investigate the suitability of amplicon-based sequencing in identifying mutations in marginally small number of samples using Ion Torrent PGM, we successfully amplified the 91 SNPs located in 69 genes out of 100 targeted SNPs across the 75 genes in 52 samples of each Horn cancer (HC) and HN (91 % coverage of targeted SNPs)
We demonstrated an efficient approach for limited number of SNP discovery and validation in targeted genomics regions in a large number of samples combining PCR amplification and next-generation sequencing technologies

Summary

Introduction

Recent advances in sequencing technologies give unrivalled opportunities to explore individual genomic landscapes and identify mutations related to cancer, a complex set of diseases characterised by both environmental and genetic contributions. The use of polymorphic markers like single-nucleotide polymorphisms (SNPs) assures to provide a comprehensive tool for analysing the mammalian genome with precise identification of genomic loci contributing to the cancer phenotype. Advances in next-generation sequencing (NGS) technologies have allowed the characterisation of SNPs at genome-wide level with high densities that were previously thought to be unachievable. NGS paved the way for the fundamental understanding of mutated genes in cancer cells, affected pathways and how these data enrich our knowledge of cancer biology (Mardis and Wilson 2009). The gene expression profiling in HC of B. indicus animals was explored with identification of SNPs in aberrantly expressed genes in our primary RNA-seq based approach

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Conclusion