Abstract
BackgroundMicrosatellite (simple sequence repeat – SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Often, large-scale genomic research projects require high-throughput computer-assisted primer design. Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. In particular, most programs lack batch primer design capability. Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded.ResultsA new web primer design program, BatchPrimer3, is developed based on Primer3. BatchPrimer3 adopted the Primer3 core program as a major primer design engine to choose the best primer pairs. A new score-based primer picking module is incorporated into BatchPrimer3 and used to pick position-restricted primers. BatchPrimer3 v1.0 implements several types of primer designs including generic primers, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. DNA sequences in FASTA format can be batch read into the program. The basic information of input sequences, as a reference of parameter setting of primer design, can be obtained by pre-analysis of sequences. The input sequences can be pre-processed and masked to exclude and/or include specific regions, or set targets for different primer design purposes as in Primer3Web and primer3Plus. A tab-delimited or Excel-formatted primer output also greatly facilitates the subsequent primer-ordering process. Thousands of primers, including wheat conserved intron-flanking primers, wheat genome-specific SNP genotyping primers, and Brachypodium SSR flanking primers in several genome projects have been designed using the program and validated in several laboratories.ConclusionBatchPrimer3 is a comprehensive web primer design program to develop different types of primers in a high-throughput manner. Additional methods of primer design can be easily integrated into future versions of BatchPrimer3. The program with source code and thousands of PCR and sequencing primers designed for wheat and Brachypodium are accessible at .
Highlights
Microsatellite and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping
Primer3 core program, a C language-written command line program, has great flexibility to optimize a number of parameters such as product size, melting temperature (Tm), GC content, primer length, 3' end stability, self complementarity, primer dimer possibility, position constraints and so forth to get the best primer pairs, and provides the potential to design different types of polymerase chain reaction (PCR) primers to meet various needs
Because only one SNP is taken as target and other SNPs are converted to one of the SNP alleles, we suggest that a separate sequence for each SNP is generated based on a reference sequence
Summary
Microsatellite (simple sequence repeat – SSR) and single nucleotide polymorphism (SNP) markers are two types of important genetic markers useful in genetic mapping and genotyping. Large-scale genomic research projects require high-throughput computer-assisted primer design Numerous such web-based or standard-alone programs for PCR primer design are available but vary in quality and functionality. Most programs lack batch primer design capability Such a high-throughput software tool for designing SSR flanking primers and SNP genotyping primers is increasingly demanded. Several other web-based or command line pipeline programs using Primer as a primer design engine have been developed [6,7,8,9,10]. Most of those webbased programs lack batch primer design capability. For many large-scale primer design projects, in addition to the requirement of suitable primer design methods, two additional features, batch input of DNA sequences and primer ordering ready output are necessary
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