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Comparative Study
. 2009 Oct 13:10:331.
doi: 10.1186/1471-2105-10-331.

ConservedPrimers 2.0: a high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery

Frank M You  1 , Naxin Huo, Yong Q Gu, Gerard R Lazo, Jan Dvorak, Olin D Anderson
Affiliations
Comparative Study

ConservedPrimers 2.0: a high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery

Frank M You et al. BMC Bioinformatics. .

Abstract

Background: In some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this _target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose.

Results: We have developed, the ConservedPrimers 2.0 pipeline, for designing intron-flanking primers for large-scale SNP discovery and marker development, and demonstrated its utility in wheat. This tool uses non-redundant wheat EST sequences, such as wheat contigs and singleton ESTs, and related genomic sequences, such as those of rice, as inputs. It aligns the ESTs to the genomic sequences to identify unique colinear exon blocks and predicts intron lengths. Intron-flanking primers are then designed based on the intron/exon information using the Primer3 core program or BatchPrimer3. Finally, a tab-delimited file containing intron-flanking primer pair sequences and their primer properties is generated for primer ordering and their PCR applications. Using this tool, 1,922 bin-mapped wheat ESTs (31.8% of the 6,045 in total) were found to have unique colinear exon blocks suitable for primer design and 1,821 primer pairs were designed from these single- or low-copy genes for PCR amplification and SNP discovery. With these primers and subsequently designed genome-specific primers, a total of 1,527 loci were found to contain one or more genome-specific SNPs.

Conclusion: The ConservedPrimers 2.0 pipeline for designing intron-flanking primers was developed and its utility demonstrated. The tool can be used for SNP discovery, genetic variation assays and marker development for any _target genome that has abundant ESTs and a related reference genome that has been fully sequenced. The ConservedPrimers 2.0 pipeline has been implemented as a command-line tool as well as a web application. Both versions are freely available at http://wheat.pw.usda.gov/demos/ConservedPrimers/.

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Figures

Figure 1
Figure 1
Schematic presentation of the intron-flanking primer design pipeline, ConservedPrimers 2.0: primer design workflow (A) and command-line pipeline programs (B).
Figure 2
Figure 2
A colinear exon block between an EST and a reference genome. (A) A colinear exon block is found if there are two or more consecutive exon matches (i.e., one or more splice sites) within a gene and if the intron length between two consecutive exons is less than 1.5 Kb. (B) If the intron length between two consecutive exons is larger than 1.5 Kb, the alignment may be split into two smaller colinear exon blocks if possible. (C) A non-unique colinear exon block is found if the same region of an EST sequence has two or more colinear exon blocks found in different locations of the reference genome. The ESTs with non-unique colinear exon blocks are excluded from conserved primer design to avoid amplifying paralogous genes.
Figure 3
Figure 3
Screenshot of the web implementation of the ConservedPrimers 2.0 pipeline.
Figure 4
Figure 4
Histograms of alignment identity (a percentage of identical matches over alignment length) (A), alignment length (B), predicted intron length (C) and number of predicted introns (D) in alignments of 1,922 wheat bin-mapped ESTs showing unique colinear exon blocks with the rice genome. Histograms were drawn using the JMP 7.0 software (SAS Institute Inc.).
Figure 5
Figure 5
Comparison between the total amplicon sequence sizes (bp) in wheat and the predicted PCR product sizes on the basis of rice genome sequence (A), and comparison between the amplicon intron length (bp) and the predicted intron length (bp) (B) based on alignments of wheat ESTs to the rice genome. A total of 888 data points from 145 homologous group 1 primer pairs and their amplicons and genomic sequences were used to draw scatter plots and histograms using the JMP 7.0 software (SAS Institute Inc.). A significant regression line was fit between the total amplicon sequence sizes in wheat and the predicted PCR product sizes (A) as well as between the amplicon intron lengths and the predicted intron lengths (B). The amplified intron lengths were determined using BLAST searches of amplified genomic sequences against their corresponding ESTs.
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