Agarwala

Camacho

Hasman

Henrik Hasman

Henrik Westh

Hülya Kaya

Inanc Birol

Kalendar

Martin Christen Frølund Thomsen

Ole Lund

Roer

Untergasser

Xavier

Abstract
               
                  Motivation
                  Designing PCR primers to target a specific selection of whole genome sequenced strains can be a long, arduous and sometimes impractical task. Such tasks would benefit greatly from an automated tool to both identify unique targets, and to validate the vast number of potential primer pairs for the targets in silico.
               
               
                  Results
                  Here we present RUCS, a program that will find PCR primer pairs and probes for the unique core sequences of a positive genome dataset complement to a negative genome dataset. The resulting primer pairs and probes are in addition to simple selection also validated through a complex in silico PCR simulation. We compared our method, which identifies the unique core sequences, against an existing tool called ssGeneFinder, and found that our method was 6.5–20 times more sensitive. We used RUCS to design primer pairs that would target a set of genomes known to contain the mcr-1 colistin resistance gene. Three of the predicted pairs were chosen for experimental validation using PCR and gel electrophoresis. All three pairs successfully produced an amplicon with the target length for the samples containing mcr-1 and no amplification products were produced for the negative samples. The novel methods presented in this manuscript can reduce the time needed to identify target sequences, and provide a quick virtual PCR validation to eliminate time wasted on ambiguously binding primers.
               
               
                  Availability and implementation
                  Source code is freely available on https://bitbucket.org/genomicepidemiology/rucs. Web service is freely available on https://cge.cbs.dtu.dk/services/RUCS.
               
               
                  Supplementary information
                  Supplementary data are available at Bioinformatics online.
               </jats:sec

Crossref

Bioinformatics

RUCS: rapid identification of PCR primers for unique core sequences

RUCS: rapid identification of PCR primers for unique core sequences

Abstract

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