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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

Primers and PCR reactions for allelic genotyping of an L1 element (a) and standard gel electrophoresis analysis of the E and G reaction products (b). a: The E reaction uses primers located in each of the L1 flanks and tests for the presence of an allele without the L1 insertion. The G reaction uses an L1-specific primer and the primer in the 3’ flank and tests for the presence of an allele bearing the L1 insertion. Primers used in the first PCR (red) are tailed (SP1 and SP2 sequences) so that the adapter (P5 and P7) and index sequences can be added in a second round PCR (green primers). b: Three possible diallelic genotypes based on the presence or absence of the E and G reaction products on an electrophoresis gel. Black filled and empty boxes represent respectively, the presence and absence of a PCR product. The gray filled boxes (marked with an asterisk) represent the (longer) product from the E reaction that is generated in the presence of an allele bearing a short L1 insertion
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Fig2: Primers and PCR reactions for allelic genotyping of an L1 element (a) and standard gel electrophoresis analysis of the E and G reaction products (b). a: The E reaction uses primers located in each of the L1 flanks and tests for the presence of an allele without the L1 insertion. The G reaction uses an L1-specific primer and the primer in the 3’ flank and tests for the presence of an allele bearing the L1 insertion. Primers used in the first PCR (red) are tailed (SP1 and SP2 sequences) so that the adapter (P5 and P7) and index sequences can be added in a second round PCR (green primers). b: Three possible diallelic genotypes based on the presence or absence of the E and G reaction products on an electrophoresis gel. Black filled and empty boxes represent respectively, the presence and absence of a PCR product. The gray filled boxes (marked with an asterisk) represent the (longer) product from the E reaction that is generated in the presence of an allele bearing a short L1 insertion

Mentions: We first applied our method to the genotyping of L1 insertions. In order to distinguish individuals with homozygous insertion alleles (“homozygous present”) from heterozygous individuals, we adapted the standard scheme requiring two PCR reactions per insertion locus (Fig. 2a). We combined this binary presence/absence read-out scheme with our read count-based method to reliably and efficiently scale up the analysis over many sites and samples. The “E” (for empty) reaction uses site-specific primers in the flanks on each side of the insertion and the “G” (for genomic) reaction uses an L1-specific primer at the 3’end of the element and the site-specific primer in the 3’ genomic flank of the insertion (Fig. 2a). On an electrophoresis gel, an allele that does not carry the L1 insertion yields a product for the E reaction and no product for the G reaction whereas an allele bearing the L1 insertion yields a product for the G reaction but generally no product for the E reaction because L1 insertions are long and prevent efficient amplification. Together the two reactions can thus differentiate the three possible diallelic genotypes (Fig. 2b). The primers for the E and G reactions are tailed with universal sequences (SP1 and SP2) to allow for the second round PCR that adds barcodes (uniquely identifying each individual) and high-throughput sequencing adapters (Fig. 2a).Fig. 2


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)

Primers and PCR reactions for allelic genotyping of an L1 element (a) and standard gel electrophoresis analysis of the E and G reaction products (b). a: The E reaction uses primers located in each of the L1 flanks and tests for the presence of an allele without the L1 insertion. The G reaction uses an L1-specific primer and the primer in the 3’ flank and tests for the presence of an allele bearing the L1 insertion. Primers used in the first PCR (red) are tailed (SP1 and SP2 sequences) so that the adapter (P5 and P7) and index sequences can be added in a second round PCR (green primers). b: Three possible diallelic genotypes based on the presence or absence of the E and G reaction products on an electrophoresis gel. Black filled and empty boxes represent respectively, the presence and absence of a PCR product. The gray filled boxes (marked with an asterisk) represent the (longer) product from the E reaction that is generated in the presence of an allele bearing a short L1 insertion
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Primers and PCR reactions for allelic genotyping of an L1 element (a) and standard gel electrophoresis analysis of the E and G reaction products (b). a: The E reaction uses primers located in each of the L1 flanks and tests for the presence of an allele without the L1 insertion. The G reaction uses an L1-specific primer and the primer in the 3’ flank and tests for the presence of an allele bearing the L1 insertion. Primers used in the first PCR (red) are tailed (SP1 and SP2 sequences) so that the adapter (P5 and P7) and index sequences can be added in a second round PCR (green primers). b: Three possible diallelic genotypes based on the presence or absence of the E and G reaction products on an electrophoresis gel. Black filled and empty boxes represent respectively, the presence and absence of a PCR product. The gray filled boxes (marked with an asterisk) represent the (longer) product from the E reaction that is generated in the presence of an allele bearing a short L1 insertion
Mentions: We first applied our method to the genotyping of L1 insertions. In order to distinguish individuals with homozygous insertion alleles (“homozygous present”) from heterozygous individuals, we adapted the standard scheme requiring two PCR reactions per insertion locus (Fig. 2a). We combined this binary presence/absence read-out scheme with our read count-based method to reliably and efficiently scale up the analysis over many sites and samples. The “E” (for empty) reaction uses site-specific primers in the flanks on each side of the insertion and the “G” (for genomic) reaction uses an L1-specific primer at the 3’end of the element and the site-specific primer in the 3’ genomic flank of the insertion (Fig. 2a). On an electrophoresis gel, an allele that does not carry the L1 insertion yields a product for the E reaction and no product for the G reaction whereas an allele bearing the L1 insertion yields a product for the G reaction but generally no product for the E reaction because L1 insertions are long and prevent efficient amplification. Together the two reactions can thus differentiate the three possible diallelic genotypes (Fig. 2b). The primers for the E and G reactions are tailed with universal sequences (SP1 and SP2) to allow for the second round PCR that adds barcodes (uniquely identifying each individual) and high-throughput sequencing adapters (Fig. 2a).Fig. 2

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