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Quality assessment parameters for EST-derived SNPs from catfish.

Wang S, Sha Z, Sonstegard TS, Liu H, Xu P, Somridhivej B, Peatman E, Kucuktas H, Liu Z - BMC Genomics (2008)

Bottom Line: However, discovery of SNPs requires genome sequencing efforts through whole genome sequencing or deep sequencing of reduced representation libraries.PCR extension appeared to be limited to a very short distance, prohibiting successful genotyping when an intron was present, a surprising finding.Application of such quality assessment measures, along with large resources of ESTs, should provide effective means for SNP identification in species where genome sequence resources are lacking.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Aquatic Genomics Unit, Auburn University, Auburn, AL 36849, USA. wangsha@auburn.edu

ABSTRACT

Background: SNPs are abundant, codominantly inherited, and sequence-tagged markers. They are highly adaptable to large-scale automated genotyping, and therefore, are most suitable for association studies and applicable to comparative genome analysis. However, discovery of SNPs requires genome sequencing efforts through whole genome sequencing or deep sequencing of reduced representation libraries. Such genome resources are not yet available for many species including catfish. A large resource of ESTs is to become available in catfish allowing identification of large number of SNPs, but reliability of EST-derived SNPs are relatively low because of sequencing errors. This project was designed to answer some of the questions relevant to quality assessment of EST-derived SNPs.

Results: wo factors were found to be most significant for validation of EST-derived SNPs: the contig size (number of sequences in the contig) and the minor allele sequence frequency. The larger the contigs were, the greater the validation rate although the validation rate was reasonably high when the contigs contain four or more EST sequences with the minor allele sequence being represented at least twice in the contigs. Sequence quality surrounding the SNP under test is also crucially important. PCR extension appeared to be limited to a very short distance, prohibiting successful genotyping when an intron was present, a surprising finding.

Conclusion: Stringent quality assessment measures should be used when working with EST-derived SNPs. In particular, contigs containing four or more ESTs should be used and the minor allele sequence should be represented at least twice. Genotyping primers should be designed from a single exon, completely avoiding introns. Application of such quality assessment measures, along with large resources of ESTs, should provide effective means for SNP identification in species where genome sequence resources are lacking.

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Schematic illustration of the effect of introns involved in SNP genotyping. In the first case, all the genotyping primers are located in the same exon nearby, leading to successful genotyping (+); in the second case (middle), one of the genotyping primers (P3 as shown) was located at the exon-intron border, causing non-base pairing that lead to failure of genotyping (-); and in the third case, even though all primers were located in exon regions. However, an intron was involved that demands PCR extension to across the intron. Apparently, the Bead array technology provide very limited extension capability, leading to genotyping failure (-) as well.
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Figure 3: Schematic illustration of the effect of introns involved in SNP genotyping. In the first case, all the genotyping primers are located in the same exon nearby, leading to successful genotyping (+); in the second case (middle), one of the genotyping primers (P3 as shown) was located at the exon-intron border, causing non-base pairing that lead to failure of genotyping (-); and in the third case, even though all primers were located in exon regions. However, an intron was involved that demands PCR extension to across the intron. Apparently, the Bead array technology provide very limited extension capability, leading to genotyping failure (-) as well.

Mentions: Selection of SNPs to allow both allele-specific and locus-specific primers to be located in a single exon is the key to achieving high success rate of SNP genotyping. We found that all tested SNP sites involving introns failed in genotyping. There seemed to be different reasons for such genotyping failures. The most notable cause is that the genotyping primers are located at exon-intron boundary, leading to non-base pairing of the primers with DNA amplified from genomic DNA (Figure 3). In addition, it appeared that the extension of the genotyping primer P1 and/or P2 to reach P3 (see Materials and Methods above) is quite limited. In cases when even both genotyping primers had a perfect match with the template DNA, genotyping failed also simply because an intron was predicted to be present between the genotyping primers (Figure 3). This is somewhat unexpected as one would expect that DNA polymerase should be able to extend easily a few hundred bases. In addition to the few tested loci, comparative gene organization analysis suggested that the vast majority of failed SNPs involved introns immediately flanking the SNP sites, further supporting the inability of genotyping when SNP is located at the exon-intron boundary or when introns are included in the extension reaction. Therefore, bioinformatics analysis using in silico comparative sequence and gene structural analysis is important when dealing with EST-derived SNPs.


Quality assessment parameters for EST-derived SNPs from catfish.

Wang S, Sha Z, Sonstegard TS, Liu H, Xu P, Somridhivej B, Peatman E, Kucuktas H, Liu Z - BMC Genomics (2008)

Schematic illustration of the effect of introns involved in SNP genotyping. In the first case, all the genotyping primers are located in the same exon nearby, leading to successful genotyping (+); in the second case (middle), one of the genotyping primers (P3 as shown) was located at the exon-intron border, causing non-base pairing that lead to failure of genotyping (-); and in the third case, even though all primers were located in exon regions. However, an intron was involved that demands PCR extension to across the intron. Apparently, the Bead array technology provide very limited extension capability, leading to genotyping failure (-) as well.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570692&req=5

Figure 3: Schematic illustration of the effect of introns involved in SNP genotyping. In the first case, all the genotyping primers are located in the same exon nearby, leading to successful genotyping (+); in the second case (middle), one of the genotyping primers (P3 as shown) was located at the exon-intron border, causing non-base pairing that lead to failure of genotyping (-); and in the third case, even though all primers were located in exon regions. However, an intron was involved that demands PCR extension to across the intron. Apparently, the Bead array technology provide very limited extension capability, leading to genotyping failure (-) as well.
Mentions: Selection of SNPs to allow both allele-specific and locus-specific primers to be located in a single exon is the key to achieving high success rate of SNP genotyping. We found that all tested SNP sites involving introns failed in genotyping. There seemed to be different reasons for such genotyping failures. The most notable cause is that the genotyping primers are located at exon-intron boundary, leading to non-base pairing of the primers with DNA amplified from genomic DNA (Figure 3). In addition, it appeared that the extension of the genotyping primer P1 and/or P2 to reach P3 (see Materials and Methods above) is quite limited. In cases when even both genotyping primers had a perfect match with the template DNA, genotyping failed also simply because an intron was predicted to be present between the genotyping primers (Figure 3). This is somewhat unexpected as one would expect that DNA polymerase should be able to extend easily a few hundred bases. In addition to the few tested loci, comparative gene organization analysis suggested that the vast majority of failed SNPs involved introns immediately flanking the SNP sites, further supporting the inability of genotyping when SNP is located at the exon-intron boundary or when introns are included in the extension reaction. Therefore, bioinformatics analysis using in silico comparative sequence and gene structural analysis is important when dealing with EST-derived SNPs.

Bottom Line: However, discovery of SNPs requires genome sequencing efforts through whole genome sequencing or deep sequencing of reduced representation libraries.PCR extension appeared to be limited to a very short distance, prohibiting successful genotyping when an intron was present, a surprising finding.Application of such quality assessment measures, along with large resources of ESTs, should provide effective means for SNP identification in species where genome sequence resources are lacking.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Aquatic Genomics Unit, Auburn University, Auburn, AL 36849, USA. wangsha@auburn.edu

ABSTRACT

Background: SNPs are abundant, codominantly inherited, and sequence-tagged markers. They are highly adaptable to large-scale automated genotyping, and therefore, are most suitable for association studies and applicable to comparative genome analysis. However, discovery of SNPs requires genome sequencing efforts through whole genome sequencing or deep sequencing of reduced representation libraries. Such genome resources are not yet available for many species including catfish. A large resource of ESTs is to become available in catfish allowing identification of large number of SNPs, but reliability of EST-derived SNPs are relatively low because of sequencing errors. This project was designed to answer some of the questions relevant to quality assessment of EST-derived SNPs.

Results: wo factors were found to be most significant for validation of EST-derived SNPs: the contig size (number of sequences in the contig) and the minor allele sequence frequency. The larger the contigs were, the greater the validation rate although the validation rate was reasonably high when the contigs contain four or more EST sequences with the minor allele sequence being represented at least twice in the contigs. Sequence quality surrounding the SNP under test is also crucially important. PCR extension appeared to be limited to a very short distance, prohibiting successful genotyping when an intron was present, a surprising finding.

Conclusion: Stringent quality assessment measures should be used when working with EST-derived SNPs. In particular, contigs containing four or more ESTs should be used and the minor allele sequence should be represented at least twice. Genotyping primers should be designed from a single exon, completely avoiding introns. Application of such quality assessment measures, along with large resources of ESTs, should provide effective means for SNP identification in species where genome sequence resources are lacking.

Show MeSH
Related in: MedlinePlus