VCF2CAPS-A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset.

Wojciech Wesołowski,Marek Szklarczyk,Beata Domnicz,Joanna Augustynowicz,Mihaela Pertea

doi:10.1371/journal.pcbi.1008980

Wojciech Wesołowski, Marek Szklarczyk + Show 3 more

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https://doi.org/10.1371/journal.pcbi.1008980

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Abstract

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

Highlights

Next-generation sequencing (NGS) technologies are powerful tools for in-depth genetic studies of even poorly characterized species
It is worth mentioning that the term cleaved amplified polymorphic sequence (CAPS) is used mostly by plant scientists while in human and animal genetics these markers are rather referred to as PCR-RFLPs–under this name they were recognized as the “gold standard” of molecular diagnostics [11]
It is a set of programs for detection of restriction polymorphisms in GFF3-formatted data and for subsequent PCR primer design. This software may not be suitable for all users and for all types of data. This is partially due to the fact that the Galaxy version of PCR Markers Tools requires setting up the Galaxy server on a local computer or in the cloud

Summary

Introduction

Next-generation sequencing (NGS) technologies are powerful tools for in-depth genetic studies of even poorly characterized species. Complexity reduction ( referred to as reduced representation) is usually achieved through application of a restriction digest during preparation of sequencing libraries, as in the case of RAD-seq (restriction site-associated DNA sequencing, [7]) or GBS (genotyping-by-sequencing, [8]) In these contexts, sequencing is typically performed using various Illumina systems of which the most powerful generate data outputs at a terabase level (https://illumina.com). PCR detection of DNA polymorphisms offers several alternatives, among them cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within amplification products Since their invention [9] CAPS markers have attracted substantial interest in genetic research, mostly due to their co-dominant character, methodological simplicity as well as ease of result interpretation [10]. It is worth mentioning that the term CAPS is used mostly by plant scientists while in human and animal genetics these markers are rather referred to as PCR-RFLPs–under this name they were recognized as the “gold standard” of molecular diagnostics [11]

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