Abstract
BackgroundBeef traceability has become mandatory in many regions of the world and is typically achieved through the use of unique numerical codes on ear tags and animal passports. DNA-based traceability uses the animal's own DNA code to identify it and the products derived from it. Using SNaPshot, a primer-extension-based method, a multiplex of 25 SNPs in a single reaction has been practiced for reducing the expense of genotyping a panel of SNPs useful for identity control.FindingsTo further decrease SNaPshot's cost, we introduced the Perl script SNPmplexViewer, which facilitates the analysis of trace files for reactions performed without the use of fluorescent size standards. SNPmplexViewer automatically aligns reference and target trace electropherograms, run with and without fluorescent size standards, respectively. SNPmplexViewer produces a modified target trace file containing a normalised trace in which the reference size standards are embedded. SNPmplexViewer also outputs aligned images of the two electropherograms together with a difference profile.ConclusionsModified trace files generated by SNPmplexViewer enable genotyping of SnaPshot reactions performed without fluorescent size standards, using common fragment-sizing software packages. SNPmplexViewer's normalised output may also improve the genotyping software's performance. Thus, SNPmplexViewer is a general free tool enabling the reduction of SNaPshot's cost as well as the fast viewing and comparing of trace electropherograms for fragment analysis. SNPmplexViewer is available at http://cowry.agri.huji.ac.il/cgi-bin/SNPmplexViewer.cgi.
Highlights
Beef traceability has become mandatory in many regions of the world and is typically achieved through the use of unique numerical codes on ear tags and animal passports
Modified trace files generated by SNPmplexViewer enable genotyping of SnaPshot reactions performed without fluorescent size standards, using common fragment-sizing software packages
Fragment analysis refers to any analysis of genetic markers which relies on variations in the length of a specific DNA sequence to indicate the presence or absence of certain marker alleles
Summary
SNPmplexViewer is based on the algorithm implemented in SeqDoC. It follows the steps of reading channel data from trace files, normalising and trimming the traces, aligning the traces and generating trace and difference images, which help the user validate the alignment. It creates a modified target trace file containing a normalised trace in which the reference size standards are embedded. By transferring the size standard information from the reference trace into the aligned target trace, a modified target trace file is formed. This modified trace allows fragment analysis of traces in the absence of size standards and reducing this analysis cost as well as improved allele calling due to the trace-normalisation process. List of abbreviations CGI, common gateway interface; SNP, single nucleotide polymorphism
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