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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

EHH analysis in the 100 kb regions around 6 selected L1 insertions (included in the 22-loci libraries) using the genotypes and phase information obtained from 40 CEU samples. P1_M_061510_1_185 (a) and P1_MEI_1280&P2_MEI_1388 (b) show clear haplotype differences between the L1-bearing allele (red) and the allele without insertion (black). The remaining insertions (c-f, corresponding to P1_M_061510_1_239, P1_M_061510_1_391, P1_M_061510_4_203, P1_M_061510_10_203, respectively) do not show clear haplotype differences between both alleles
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Fig5: EHH analysis in the 100 kb regions around 6 selected L1 insertions (included in the 22-loci libraries) using the genotypes and phase information obtained from 40 CEU samples. P1_M_061510_1_185 (a) and P1_MEI_1280&P2_MEI_1388 (b) show clear haplotype differences between the L1-bearing allele (red) and the allele without insertion (black). The remaining insertions (c-f, corresponding to P1_M_061510_1_239, P1_M_061510_1_391, P1_M_061510_4_203, P1_M_061510_10_203, respectively) do not show clear haplotype differences between both alleles

Mentions: We used the phase information obtained here to analyze the haplotypic structure around a small set of L1 insertions. Haplotypic structures can provide useful information on the evolutionary dynamics of genetic variants: We previously detected significant extended haplotype homozygosity (EHH) around particular L1 insertions, compatible with the signature left by recent and rapid positive selection events. To ensure a high genotyping accuracy, we had genotyped these L1s using individual, site-specific PCRs and gel electrophoresis analysis. Because this standard genotyping approach cannot yield L1 phases, however, we had to restrict our analysis to homozygous samples only. Here, we repeated the EHH analysis around 7 L1 insertions that we included in the 22-loci experiment and that had been previously analyzed. We used L1 genotypes and phase information obtained with our sequencing-based method, resulting in the additional inclusion of phased heterozygous samples in the analysis. We included the 24 samples assayed for the validation experiment and performed read count-based genotyping on 16 additional samples to obtain a cohort of 40 samples. For each locus, the phase information allowed us to include between 1 and 15 additional (heterozygous) samples in the analysis (depending on the number of heterozygous L1 samples containing at least one heterozygous SNP in the L1 flanking region). Out of the 7 L1 insertions assayed here, 6 were successfully genotyped and phased and two showed strong differences in EHH signals obtained for the alleles with (red) and without (black) L1 insertion, reflecting haplotypic differences on both alleles (Fig. 5). They indeed correspond to 2 of the 3 L1s identified in our previous study (the third L1 insertion was not included in the 22-loci libraries and hence could not be analyzed) and that might have been under positive selection in recent human evolution.Fig. 5


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)

EHH analysis in the 100 kb regions around 6 selected L1 insertions (included in the 22-loci libraries) using the genotypes and phase information obtained from 40 CEU samples. P1_M_061510_1_185 (a) and P1_MEI_1280&P2_MEI_1388 (b) show clear haplotype differences between the L1-bearing allele (red) and the allele without insertion (black). The remaining insertions (c-f, corresponding to P1_M_061510_1_239, P1_M_061510_1_391, P1_M_061510_4_203, P1_M_061510_10_203, respectively) do not show clear haplotype differences between both alleles
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: EHH analysis in the 100 kb regions around 6 selected L1 insertions (included in the 22-loci libraries) using the genotypes and phase information obtained from 40 CEU samples. P1_M_061510_1_185 (a) and P1_MEI_1280&P2_MEI_1388 (b) show clear haplotype differences between the L1-bearing allele (red) and the allele without insertion (black). The remaining insertions (c-f, corresponding to P1_M_061510_1_239, P1_M_061510_1_391, P1_M_061510_4_203, P1_M_061510_10_203, respectively) do not show clear haplotype differences between both alleles
Mentions: We used the phase information obtained here to analyze the haplotypic structure around a small set of L1 insertions. Haplotypic structures can provide useful information on the evolutionary dynamics of genetic variants: We previously detected significant extended haplotype homozygosity (EHH) around particular L1 insertions, compatible with the signature left by recent and rapid positive selection events. To ensure a high genotyping accuracy, we had genotyped these L1s using individual, site-specific PCRs and gel electrophoresis analysis. Because this standard genotyping approach cannot yield L1 phases, however, we had to restrict our analysis to homozygous samples only. Here, we repeated the EHH analysis around 7 L1 insertions that we included in the 22-loci experiment and that had been previously analyzed. We used L1 genotypes and phase information obtained with our sequencing-based method, resulting in the additional inclusion of phased heterozygous samples in the analysis. We included the 24 samples assayed for the validation experiment and performed read count-based genotyping on 16 additional samples to obtain a cohort of 40 samples. For each locus, the phase information allowed us to include between 1 and 15 additional (heterozygous) samples in the analysis (depending on the number of heterozygous L1 samples containing at least one heterozygous SNP in the L1 flanking region). Out of the 7 L1 insertions assayed here, 6 were successfully genotyped and phased and two showed strong differences in EHH signals obtained for the alleles with (red) and without (black) L1 insertion, reflecting haplotypic differences on both alleles (Fig. 5). They indeed correspond to 2 of the 3 L1s identified in our previous study (the third L1 insertion was not included in the 22-loci libraries and hence could not be analyzed) and that might have been under positive selection in recent human evolution.Fig. 5

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