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High throughput sequencing in mice: a platform comparison identifies a preponderance of cryptic SNPs.

Walter NA, Bottomly D, Laderas T, Mooney MA, Darakjian P, Searles RP, Harrington CA, McWeeney SK, Hitzemann R, Buck KJ - BMC Genomics (2009)

Bottom Line: Polymorphisms result in a high incidence of false positive and false negative results in hybridization based analyses and hinder the identification of the true variation underlying genetically determined differences in physiology and behavior.Using the same templates on both platforms, we compared realignments and single nucleotide polymorphism (SNP) detection with an 80 fold average read depth across platforms and samples.Furthermore, we confirmed 40 missense SNPs and discovered 36 new missense SNPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research and Development Service, Portland VA Medical Center, Portland, OR, USA. waltern@ohsu.edu

ABSTRACT

Background: Allelic variation is the cornerstone of genetically determined differences in gene expression, gene product structure, physiology, and behavior. However, allelic variation, particularly cryptic (unknown or not annotated) variation, is problematic for follow up analyses. Polymorphisms result in a high incidence of false positive and false negative results in hybridization based analyses and hinder the identification of the true variation underlying genetically determined differences in physiology and behavior. Given the proliferation of mouse genetic models (e.g., knockout models, selectively bred lines, heterogeneous stocks derived from standard inbred strains and wild mice) and the wealth of gene expression microarray and phenotypic studies using genetic models, the impact of naturally-occurring polymorphisms on these data is critical. With the advent of next-generation, high-throughput sequencing, we are now in a position to determine to what extent polymorphisms are currently cryptic in such models and their impact on downstream analyses.

Results: We sequenced the two most commonly used inbred mouse strains, DBA/2J and C57BL/6J, across a region of chromosome 1 (171.6 - 174.6 megabases) using two next generation high-throughput sequencing platforms: Applied Biosystems (SOLiD) and Illumina (Genome Analyzer). Using the same templates on both platforms, we compared realignments and single nucleotide polymorphism (SNP) detection with an 80 fold average read depth across platforms and samples. While public datasets currently annotate 4,527 SNPs between the two strains in this interval, thorough high-throughput sequencing identified a total of 11,824 SNPs in the interval, including 7,663 new SNPs. Furthermore, we confirmed 40 missense SNPs and discovered 36 new missense SNPs.

Conclusion: Comparisons utilizing even two of the best characterized mouse genetic models, DBA/2J and C57BL/6J, indicate that more than half of naturally-occurring SNPs remain cryptic. The magnitude of this problem is compounded when using more divergent or poorly annotated genetic models. This warrants full genomic sequencing of the mouse strains used as genetic models.

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BAC sequencing coverage. B6 strain RPCI-23 BACs used for genomic sequencing are denoted as white boxes. D2 strain MM_DBa BACs are indicated as black boxes. Each set of BACs is assembled as an overlapping contig. The 3 Mb (171.6–174.6 Mb) region used for comparisons is bracketed in red.
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Figure 1: BAC sequencing coverage. B6 strain RPCI-23 BACs used for genomic sequencing are denoted as white boxes. D2 strain MM_DBa BACs are indicated as black boxes. Each set of BACs is assembled as an overlapping contig. The 3 Mb (171.6–174.6 Mb) region used for comparisons is bracketed in red.

Mentions: Using 32 PCR probes spanning the 171.6 – 174.6 Mb interval of chromosome 1, we screened a commercially available D2 strain bacterial artificial chromosome (BAC) library, MM_DBa. End-sequencing of the BACs identified by these PCR probes allowed us to assemble a minimal overlapping contig of 27 D2 BACs (Figure 1). The resulting D2 BAC contig spanned a total of 3.1 Mb from 171,509,721 – 174,625,201 bp on chromosome 1 (Build 37). Contig sequence data from the SOLiD and Genome Anlayzer platforms were assembled via realignment to the Ensembl reference sequence [7] and covered the region surveyed without any large gaps.


High throughput sequencing in mice: a platform comparison identifies a preponderance of cryptic SNPs.

Walter NA, Bottomly D, Laderas T, Mooney MA, Darakjian P, Searles RP, Harrington CA, McWeeney SK, Hitzemann R, Buck KJ - BMC Genomics (2009)

BAC sequencing coverage. B6 strain RPCI-23 BACs used for genomic sequencing are denoted as white boxes. D2 strain MM_DBa BACs are indicated as black boxes. Each set of BACs is assembled as an overlapping contig. The 3 Mb (171.6–174.6 Mb) region used for comparisons is bracketed in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743714&req=5

Figure 1: BAC sequencing coverage. B6 strain RPCI-23 BACs used for genomic sequencing are denoted as white boxes. D2 strain MM_DBa BACs are indicated as black boxes. Each set of BACs is assembled as an overlapping contig. The 3 Mb (171.6–174.6 Mb) region used for comparisons is bracketed in red.
Mentions: Using 32 PCR probes spanning the 171.6 – 174.6 Mb interval of chromosome 1, we screened a commercially available D2 strain bacterial artificial chromosome (BAC) library, MM_DBa. End-sequencing of the BACs identified by these PCR probes allowed us to assemble a minimal overlapping contig of 27 D2 BACs (Figure 1). The resulting D2 BAC contig spanned a total of 3.1 Mb from 171,509,721 – 174,625,201 bp on chromosome 1 (Build 37). Contig sequence data from the SOLiD and Genome Anlayzer platforms were assembled via realignment to the Ensembl reference sequence [7] and covered the region surveyed without any large gaps.

Bottom Line: Polymorphisms result in a high incidence of false positive and false negative results in hybridization based analyses and hinder the identification of the true variation underlying genetically determined differences in physiology and behavior.Using the same templates on both platforms, we compared realignments and single nucleotide polymorphism (SNP) detection with an 80 fold average read depth across platforms and samples.Furthermore, we confirmed 40 missense SNPs and discovered 36 new missense SNPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research and Development Service, Portland VA Medical Center, Portland, OR, USA. waltern@ohsu.edu

ABSTRACT

Background: Allelic variation is the cornerstone of genetically determined differences in gene expression, gene product structure, physiology, and behavior. However, allelic variation, particularly cryptic (unknown or not annotated) variation, is problematic for follow up analyses. Polymorphisms result in a high incidence of false positive and false negative results in hybridization based analyses and hinder the identification of the true variation underlying genetically determined differences in physiology and behavior. Given the proliferation of mouse genetic models (e.g., knockout models, selectively bred lines, heterogeneous stocks derived from standard inbred strains and wild mice) and the wealth of gene expression microarray and phenotypic studies using genetic models, the impact of naturally-occurring polymorphisms on these data is critical. With the advent of next-generation, high-throughput sequencing, we are now in a position to determine to what extent polymorphisms are currently cryptic in such models and their impact on downstream analyses.

Results: We sequenced the two most commonly used inbred mouse strains, DBA/2J and C57BL/6J, across a region of chromosome 1 (171.6 - 174.6 megabases) using two next generation high-throughput sequencing platforms: Applied Biosystems (SOLiD) and Illumina (Genome Analyzer). Using the same templates on both platforms, we compared realignments and single nucleotide polymorphism (SNP) detection with an 80 fold average read depth across platforms and samples. While public datasets currently annotate 4,527 SNPs between the two strains in this interval, thorough high-throughput sequencing identified a total of 11,824 SNPs in the interval, including 7,663 new SNPs. Furthermore, we confirmed 40 missense SNPs and discovered 36 new missense SNPs.

Conclusion: Comparisons utilizing even two of the best characterized mouse genetic models, DBA/2J and C57BL/6J, indicate that more than half of naturally-occurring SNPs remain cryptic. The magnitude of this problem is compounded when using more divergent or poorly annotated genetic models. This warrants full genomic sequencing of the mouse strains used as genetic models.

Show MeSH
Related in: MedlinePlus