SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development

Published online January 2, 2004 Nucleic Acids Research, 2004, Vol. 32, No. 1 e5 DOI: 10.1093/nar/gnh006 SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development Thomas Thiel, Raja Kota, Ivo Grosse, Nils Stein and Andreas Graner* Institute for Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany Received August 12, 2003; Revised and Accepted November 14, 2003 ABSTRACT INTRODUCTION Single nucleotide polymorphisms (SNPs) are the most frequent form of DNA variation in the genome (1). Because of their abundance, the exploitation of SNPs for marker assays has the potential to provide answers to a large number of important biological, genetic, pharmacological and medical questions. The identi®cation of SNPs has progressed remarkably over the last several years and multiple assays have been devised (2±5). However, most of these assays require expensive and specialized equipment and chemicals for analysis. Hence, there is a need for simple and accurate genotyping assays that can be implemented in laboratories that do not have access to sophisticated equipment. A solution to this problem is the detection of a SNP site by an appropriate restriction endonuclease whose recognition sequence has been altered or introduced by the SNP. In combination with a PCR assay, the corresponding SNP can be analysed as a cleaved ampli®ed polymorphic sequence (CAPS) marker (6). The MATERIALS AND METHODS Description of SNP2CAPS Two input ®les that contain data about the sequence alignments and the restriction enzymes are required for SNP2CAPS. The ®rst input ®le is a modi®ed FASTA formatted ®le that stores one or more multiple alignments of sequences of different accessions. In order to ensure compatibility with existing alignment tools, 15 additional multiple alignment formats (e.g. ClustalW, MSF and MEME) can be imported using the AlignIO handler of the BioPerl module v1.2 (http://bioperl.org/). The second input ®le contains data on the restriction enzymes that can be downloaded in different *To whom correspondence should be addressed. Tel: +49 39482 5521; Fax: +49 39482 5155; Email: The authors wish it to be known that, in their opinion, the ®rst two authors should be regarded as joint First Authors Nucleic Acids Research, Vol. 32 No. 1 ã Oxford University Press 2004; all rights reserved With the in¯ux of various SNP genotyping assays in recent years, there has been a need for an assay that is robust, yet cost effective, and could be performed using standard gel-based procedures. In this context, CAPS markers have been shown to meet these criteria. However, converting SNPs to CAPS markers can be a dif®cult process if done manually. In order to address this problem, we describe a computer program, SNP2CAPS, that facilitates the computational conversion of SNP markers into CAPS markers. 413 multiple aligned sequences derived from barley ESTs were analysed for the presence of polymorphisms in 235 distinct restriction sites. 282 (90%) of 314 alignments that contain sequence variation due to SNPs and InDels revealed at least one polymorphic restriction site. After reducing the number of restriction enzymes from 235 to 10, 31% of the polymorphic sites could still be detected. In order to demonstrate the usefulness of this tool for marker development, we experimentally validated some of the results predicted by SNP2CAPS. costs of a CAPS assay is generally low, especially when it relies on commonly used restriction enzymes. In order to facilitate a computational conversion of SNPs into CAPS markers, a program called dCAPS Finder 2.0 was previously developed (7). The dCAPS Finder program works on the principle of designing mismatched PCR primers that would create or remove a restriction recognition site in the analysed SNP. The conversion of SNP sites into CAPS markers by the arti®cial introduction of restriction sites involves the creation of mismatched primers, whose successful application is not always trivial depending on the number, positions and types of mismatches. We present a computer program named SNP2CAPS that works in a different manner. A simple algorithm involves the screening of multiply aligned sequences for restriction sites followed by a selection pipeline that allows the deduction of CAPS candidates by the identi®cation of putative alternative restriction patterns. It should be noted that in this algorithm any primer pair ¯anking the SNP site may be suited for CAPS marker analysis. In order to evaluate the ef®cacy of SNP2CAPS, a set of 3045 sequences derived from eight barley accessions based on 413 expressed sequence tags (ESTs) was used and analysed in terms of (i) the potential number of SNP markers that can be converted into CAPS markers taking into account all commercially available restriction enzymes and (ii) the number of CAPS markers that can be typed if only the 10 most commonly used enzymes are considered. To investigate the accuracy of this tool, 14 EST-based SNP markers have been experimentally validated. e5 Nucleic Acids Research, 2004, Vol. 32, No. 1 Data sets Sequencing efforts resulted in a set of 413 partially sequenced genes spanning a total of 153 kb. On average, each locus had a length of 370 bp. 3045 sequences were obtained by sequencing three to eight barley accessions per gene locus, resulting in a total of 1.13 Mb. Sequences were aligned using the ClustalW program (8). For computational restriction analysis the GCG format data ®le from the REBASE database (version 304, March 24, 2003) was used, comprising information on 645 type II restriction enzymes. In the present study, diagnostic restriction was investigated using (i) a total of 235 enzymes that are PAGE 2 OF 5 Figure 1. Illustration of possible scenarios at an EcoRI recognition site (GAATTC) between two aligned sequences. (a±d) Four different types that are recognized by the program algorithm: (a) class I, CAPS candidates; (b) class II, CAPS candidates containing N; (c) class III, false positive candidates; (d) class IV, no restriction site polymorphisms (due to no or uniform restriction patterns). (e and f) The role of insertions/deletions for the analysis of CAPS marker candidates: (e) deletion of the restriction site; (f) insertion at a restriction site. non-isoschizomeric and commercially available and (ii) a subset of the following 10 enzymes that are widely used in daily bench work: the 4 bp cutters AluI, HpaII, MseI and RsaI and the 6 bp cutters BamHI, DraI, EcoRI, EcoRV, HindIII and XbaI. Plant material For experimental veri®cation of the SNP2CAPS results a genotype set of seven barley (Hordeum vulgare ssp. vulgare) accessions was used, namely the winter barley cultivars `Igri' and `Franka', the spring barley cultivars `Steptoe', `Morex' and `Barke', the genetic stocks `Oregon Wolfe Rec' and `Oregon Wolfe Dom' and accession HOR11508 of wild barley (H.vulgare ssp. spontaneum). For these eight accessions genomic DNA was extracted fr (...truncated)