Dataset Information

SNP discovery in swine by reduced representation and high throughput pyrosequencing.

ABSTRACT: BACKGROUND: Relatively little information is available for sequence variation in the pig. We previously used a combination of short read (25 base pair) high-throughput sequencing and reduced genomic representation to discover > 60,000 single nucleotide polymorphisms (SNP) in cattle, but the current lack of complete genome sequence limits this approach in swine. Longer-read pyrosequencing-based technologies have the potential to overcome this limitation by providing sufficient flanking sequence information for assay design. Swine SNP were discovered in the present study using a reduced representation of 450 base pair (bp) porcine genomic fragments (approximately 4% of the swine genome) prepared from a pool of 26 animals relevant to current pork production, and a GS-FLX instrument producing 240 bp reads. RESULTS: Approximately 5 million sequence reads were collected and assembled into contigs having an overall observed depth of 7.65-fold coverage. The approximate minor allele frequency was estimated from the number of observations of the alternate alleles. The average coverage at the SNPs was 12.6-fold. This approach identified 115,572 SNPs in 47,830 contigs. Comparison to partial swine genome draft sequence indicated 49,879 SNP (43%) and 22,045 contigs (46%) mapped to a position on a sequenced pig chromosome and the distribution was essentially random. A sample of 176 putative SNPs was examined and 168 (95.5%) were confirmed to have segregating alleles; the correlation of the observed minor allele frequency (MAF) to that predicted from the sequence data was 0.58. CONCLUSION: The process was an efficient means to identify a large number of porcine SNP having high validation rate to be used in an ongoing international collaboration to produce a highly parallel genotyping assay for swine. By using a conservative approach, a robust group of SNPs were detected with greater confidence and relatively high MAF that should be suitable for genotyping in a wide variety of commercial populations.

SUBMITTER: Wiedmann RT

PROVIDER: S-EPMC2612698 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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SNP discovery in swine by reduced representation and high throughput pyrosequencing.

Wiedmann Ralph T RT Smith Timothy P L TP Nonneman Dan J DJ

BMC genetics 20081204

<h4>Background</h4>Relatively little information is available for sequence variation in the pig. We previously used a combination of short read (25 base pair) high-throughput sequencing and reduced genomic representation to discover > 60,000 single nucleotide polymorphisms (SNP) in cattle, but the current lack of complete genome sequence limits this approach in swine. Longer-read pyrosequencing-based technologies have the potential to overcome this limitation by providing sufficient flanking seq ...[more]

PMID: 19055830

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Project description:BackgroundSingle nucleotide polymorphisms (SNPs) as defined here are single base sequence changes or short insertion/deletions between or within individuals of a given species. As a result of their abundance and the availability of high throughput analysis technologies SNP markers have begun to replace other traditional markers such as restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs or microsatellite) markers for fine mapping and association studies in several species. For SNP discovery from chromatogram data, several bioinformatics programs have to be combined to generate an analysis pipeline. Results have to be stored in a relational database to facilitate interrogation through queries or to generate data for further analyses such as determination of linkage disequilibrium and identification of common haplotypes. Although these tasks are routinely performed by several groups, an integrated open source SNP discovery pipeline that can be easily adapted by new groups interested in SNP marker development is currently unavailable.ResultsWe developed SNP-PHAGE (SNP discovery Pipeline with additional features for identification of common haplotypes within a sequence tagged site (Haplotype Analysis) and GenBank (-dbSNP) submissions. This tool was applied for analyzing sequence traces from diverse soybean genotypes to discover over 10,000 SNPs. This package was developed on UNIX/Linux platform, written in Perl and uses a MySQL database. Scripts to generate a user-friendly web interface are also provided with common queries for preliminary data analysis. A machine learning tool developed by this group for increasing the efficiency of SNP discovery is integrated as a part of this package as an optional feature. The SNP-PHAGE package is being made available open source at http://bfgl.anri.barc.usda.gov/ML/snp-phage/.ConclusionSNP-PHAGE provides a bioinformatics solution for high throughput SNP discovery, identification of common haplotypes within an amplicon, and GenBank (dbSNP) submissions. SNP selection and visualization are aided through a user-friendly web interface. This tool is useful for analyzing sequence tagged sites (STSs) of genomic sequences, and this software can serve as a starting point for groups interested in developing SNP markers.

Project description:BackgroundWith the advance of new massively parallel genotyping technologies, quantitative trait loci (QTL) fine mapping and map-based cloning become more achievable in identifying genes for important and complex traits. Development of high-density genetic markers in the QTL regions of specific mapping populations is essential for fine-mapping and map-based cloning of economically important genes. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation existing between any diverse genotypes that are usually used for QTL mapping studies. The massively parallel sequencing technologies (Roche GS/454, Illumina GA/Solexa, and ABI/SOLiD), have been widely applied to identify genome-wide sequence variations. However, it is still remains unclear whether sequence data at a low sequencing depth are enough to detect the variations existing in any QTL regions of interest in a crop genome, and how to prepare sequencing samples for a complex genome such as soybean. Therefore, with the aims of identifying SNP markers in a cost effective way for fine-mapping several QTL regions, and testing the validation rate of the putative SNPs predicted with Solexa short sequence reads at a low sequencing depth, we evaluated a pooled DNA fragment reduced representation library and SNP detection methods applied to short read sequences generated by Solexa high-throughput sequencing technology.ResultsA total of 39,022 putative SNPs were identified by the Illumina/Solexa sequencing system using a reduced representation DNA library of two parental lines of a mapping population. The validation rates of these putative SNPs predicted with low and high stringency were 72% and 85%, respectively. One hundred sixty four SNP markers resulted from the validation of putative SNPs and have been selectively chosen to target a known QTL, thereby increasing the marker density of the targeted region to one marker per 42 K bp.ConclusionsWe have demonstrated how to quickly identify large numbers of SNPs for fine mapping of QTL regions by applying massively parallel sequencing combined with genome complexity reduction techniques. This SNP discovery approach is more efficient for targeting multiple QTL regions in a same genetic population, which can be applied to other crops.

Project description:BACKGROUND: Flax (Linum usitatissimum L.) is a significant fibre and oilseed crop. Current flax molecular markers, including isozymes, RAPDs, AFLPs and SSRs are of limited use in the construction of high density linkage maps and for association mapping applications due to factors such as low reproducibility, intense labour requirements and/or limited numbers. We report here on the use of a reduced representation library strategy combined with next generation Illumina sequencing for rapid and large scale discovery of SNPs in eight flax genotypes. SNP discovery was performed through in silico analysis of the sequencing data against the whole genome shotgun sequence assembly of flax genotype CDC Bethune. Genotyping-by-sequencing of an F6-derived recombinant inbred line population provided validation of the SNPs. RESULTS: Reduced representation libraries of eight flax genotypes were sequenced on the Illumina sequencing platform resulting in sequence coverage ranging from 4.33 to 15.64X (genome equivalents). Depending on the relatedness of the genotypes and the number and length of the reads, between 78% and 93% of the reads mapped onto the CDC Bethune whole genome shotgun sequence assembly. A total of 55,465 SNPs were discovered with the largest number of SNPs belonging to the genotypes with the highest mapping coverage percentage. Approximately 84% of the SNPs discovered were identified in a single genotype, 13% were shared between any two genotypes and the remaining 3% in three or more. Nearly a quarter of the SNPs were found in genic regions. A total of 4,706 out of 4,863 SNPs discovered in Macbeth were validated using genotyping-by-sequencing of 96 F6 individuals from a recombinant inbred line population derived from a cross between CDC Bethune and Macbeth, corresponding to a validation rate of 96.8%. CONCLUSIONS: Next generation sequencing of reduced representation libraries was successfully implemented for genome-wide SNP discovery from flax. The genotyping-by-sequencing approach proved to be efficient for validation. The SNP resources generated in this work will assist in generating high density maps of flax and facilitate QTL discovery, marker-assisted selection, phylogenetic analyses, association mapping and anchoring of the whole genome shotgun sequence.

Project description:BackgroundSingle nucleotide polymorphisms (SNP) have been applied as important molecular markers in genetics and breeding studies. The rapid advance of next generation sequencing (NGS) provides a high-throughput means of SNP discovery. However, SNP development is limited by the availability of reliable SNP discovery methods. Especially, the optimum assembler and SNP caller for accurate SNP prediction from next generation sequencing data are not known.ResultsHerein we performed SNP prediction based on RNA-seq data of peach and mandarin peel tissue under a comprehensive comparison of two paired-end read lengths (125 bp and 150 bp), five assemblers (Trinity, IDBA, oases, SOAPdenovo, Trans-abyss) and two SNP callers (GATK and GBS). The predicted SNPs were compared with the authentic SNPs identified via PCR amplification followed by gene cloning and sequencing procedures. A total of 40 and 240 authentic SNPs were presented in five anthocyanin biosynthesis related genes in peach and in nine carotenogenic genes in mandarin. Putative SNPs predicted from the same RNA-seq data with different strategies led to quite divergent results. The rate of false positive SNPs was significantly lower when the paired-end read length was 150 bp compared with 125 bp. Trinity was superior to the other four assemblers and GATK was substantially superior to GBS due to a low rate of missing authentic SNPs. The combination of assembler Trinity, SNP caller GATK, and the paired-end read length 150 bp had the best performance in SNP discovery with 100% accuracy both in peach and in mandarin cases. This strategy was applied to the characterization of SNPs in peach and mandarin transcriptomes.ConclusionsThrough comparison of authentic SNPs obtained by PCR cloning strategy and putative SNPs predicted from different combinations of five assemblers, two SNP callers, and two paired-end read lengths, we provided a reliable and efficient strategy, Trinity-GATK with 150 bp paired-end read length, for SNP discovery from RNA-seq data. This strategy discovered SNP at 100% accuracy in peach and mandarin cases and might be applicable to a wide range of plants and other organisms.

Project description:BACKGROUND: Benefits from high-throughput sequencing using 454 pyrosequencing technology may be most apparent for species with high societal or economic value but few genomic resources. Rapid means of gene sequence and SNP discovery using this novel sequencing technology provide a set of baseline tools for genome-level research. However, it is questionable how effective the sequencing of large numbers of short reads for species with essentially no prior gene sequence information will support contig assemblies and sequence annotation. RESULTS: With the purpose of generating the first broad survey of gene sequences in Eucalyptus grandis, the most widely planted hardwood tree species, we used 454 technology to sequence and assemble 148 Mbp of expressed sequences (EST). EST sequences were generated from a normalized cDNA pool comprised of multiple tissues and genotypes, promoting discovery of homologues to almost half of Arabidopsis genes, and a comprehensive survey of allelic variation in the transcriptome. By aligning the sequencing reads from multiple genotypes we detected 23,742 SNPs, 83% of which were validated in a sample. Genome-wide nucleotide diversity was estimated for 2,392 contigs using a modified theta (theta) parameter, adapted for measuring genetic diversity from polymorphisms detected by randomly sequencing a multi-genotype cDNA pool. Diversity estimates in non-synonymous nucleotides were on average 4x smaller than in synonymous, suggesting purifying selection. Non-synonymous to synonymous substitutions (Ka/Ks) among 2,001 contigs averaged 0.30 and was skewed to the right, further supporting that most genes are under purifying selection. Comparison of these estimates among contigs identified major functional classes of genes under purifying and diversifying selection in agreement with previous researches. CONCLUSION: In providing an abundance of foundational transcript sequences where limited prior genomic information existed, this work created part of the foundation for the annotation of the E. grandis genome that is being sequenced by the US Department of Energy. In addition we demonstrated that SNPs sampled in large-scale with 454 pyrosequencing can be used to detect evolutionary signatures among genes, providing one of the first genome-wide assessments of nucleotide diversity and Ka/Ks for a non-model plant species.

Dataset Information

SNP discovery in swine by reduced representation and high throughput pyrosequencing.

Publications

SNP discovery in swine by reduced representation and high throughput pyrosequencing.

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