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, 32 (1), e5

SNP2CAPS: A SNP and INDEL Analysis Tool for CAPS Marker Development

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SNP2CAPS: A SNP and INDEL Analysis Tool for CAPS Marker Development

Thomas Thiel et al. Nucleic Acids Res.

Abstract

With the influx 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 difficult 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.

Figures

Figure 1
Figure 1
Illustration of possible scenarios at an EcoRI recognition site (G↑AATTC) 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.
Figure 2
Figure 2
Screenshot of SNP2CAPS. The screenshot shows the output of the results after the screening of SNP marker GBS0734 with 10 selected restriction enzymes.
Figure 3
Figure 3
Conversion of the barley SNP marker GBS0734 into an EcoRV CAPS marker. (a) Relevant part of the multiple sequence alignment of SNP marker GBS0734. The recognition site of EcoRV (GAT↑ATC) is affected by one SNP at position 151 (C→T transition). ‘Igri’ (I), ‘Franka’ (F), ‘Steptoe’ (S), ‘Morex’ (M), ‘Dom’ (D), ‘Rec’ (R), ‘Barke’ (B) and H.vulgare ssp. spontaneum (Sp) are different barley accessions used in this study. (b) Gel electrophoretic separation of PCR products (left undigested, right after EcoRV digestion). As predicted by SNP2CAPS, the restriction enzyme EcoRV cuts PCR fragments of ‘Igri’, ‘Franka’, ‘Morex’ and ‘Rec’ into two fragments of the predicted sizes (148 and 196 bp), whereas ‘Steptoe’, ‘Dom’, ‘Barke’ and H.vulgare ssp. spontaneum display the undigested PCR product (344 bp). Relevant fragment sizes (bp) are denoted on the right side.

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