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Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants.

Kuhn A, Ong YM, Quake SR, Burkholder WF - BMC Genomics (2015)

Bottom Line: Like other structural variants, transposable element insertions can be highly polymorphic across individuals.Their functional impact, however, remains poorly understood.This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

View Article: PubMed Central - PubMed

Affiliation: Microfluidics Systems Biology Lab, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Proteos Building, Room #03-04, 61 Biopolis Drive, Singapore, 138673, Singapore. alexandre.m.kuhn@gmail.com.

ABSTRACT

Background: Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed.

Results: We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate.

Conclusions: This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

No MeSH data available.


Related in: MedlinePlus

Principle of count-based genotyping of structural variation (a) and workflow of a genotyping experiment (b). a: Genotyping of three polymorphic TE insertion loci. Gray and dotted boxes represent present and absent insertions, respectively. Blue arrows represent PCR primers and orange lines depict sequencing reads
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Fig1: Principle of count-based genotyping of structural variation (a) and workflow of a genotyping experiment (b). a: Genotyping of three polymorphic TE insertion loci. Gray and dotted boxes represent present and absent insertions, respectively. Blue arrows represent PCR primers and orange lines depict sequencing reads

Mentions: We describe the principle of the method as applied to the genotyping of TE insertions (Fig. 1a). A set of TE loci is assayed by multiplex PCR amplifications targeting the junctions between TEs and their flanking genomic insertion sites. High multiplexing can be achieved by various (possibly combined) methods including the careful design of primers that allow for multiple PCR reactions in the same reagent volume and parallelization of PCR reactions using droplet or microfluidic technology. Amplicon libraries are sequenced, and reads matching the targeted locations are counted: a high number of specific reads indicates that the corresponding PCR reaction amplified the targeted TE junction and that the TE was present whereas the absence of specific reads (or the presence of a small number of cross-contaminating reads) indicates that the TE was absent.Fig. 1


Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants.

Kuhn A, Ong YM, Quake SR, Burkholder WF - BMC Genomics (2015)

Principle of count-based genotyping of structural variation (a) and workflow of a genotyping experiment (b). a: Genotyping of three polymorphic TE insertion loci. Gray and dotted boxes represent present and absent insertions, respectively. Blue arrows represent PCR primers and orange lines depict sequencing reads
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494700&req=5

Fig1: Principle of count-based genotyping of structural variation (a) and workflow of a genotyping experiment (b). a: Genotyping of three polymorphic TE insertion loci. Gray and dotted boxes represent present and absent insertions, respectively. Blue arrows represent PCR primers and orange lines depict sequencing reads
Mentions: We describe the principle of the method as applied to the genotyping of TE insertions (Fig. 1a). A set of TE loci is assayed by multiplex PCR amplifications targeting the junctions between TEs and their flanking genomic insertion sites. High multiplexing can be achieved by various (possibly combined) methods including the careful design of primers that allow for multiple PCR reactions in the same reagent volume and parallelization of PCR reactions using droplet or microfluidic technology. Amplicon libraries are sequenced, and reads matching the targeted locations are counted: a high number of specific reads indicates that the corresponding PCR reaction amplified the targeted TE junction and that the TE was present whereas the absence of specific reads (or the presence of a small number of cross-contaminating reads) indicates that the TE was absent.Fig. 1

Bottom Line: Like other structural variants, transposable element insertions can be highly polymorphic across individuals.Their functional impact, however, remains poorly understood.This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

View Article: PubMed Central - PubMed

Affiliation: Microfluidics Systems Biology Lab, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Proteos Building, Room #03-04, 61 Biopolis Drive, Singapore, 138673, Singapore. alexandre.m.kuhn@gmail.com.

ABSTRACT

Background: Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed.

Results: We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate.

Conclusions: This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

No MeSH data available.


Related in: MedlinePlus