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Next-generation sequencing-based detection of germline L1-mediated transductions.

Tica J, Lee E, Untergasser A, Meiers S, Garfield DA, Gokcumen O, Furlong EE, Park PJ, Stütz AM, Korbel JO - BMC Genomics (2016)

Bottom Line: We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome.We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species.By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Genome Biology Unit, 69117, Heidelberg, Germany.

ABSTRACT

Background: While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking.

Results: Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome. We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species.

Conclusions: By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.

No MeSH data available.


Related in: MedlinePlus

Pacific Biosciences (a) and Oxford Nanopore MinION (b) long read verification of L1-mediated transduction insertions. aLeft panel: alignment dotplot – surrounding reference genome sequence for the human chr4:104210671-104214687 region shown on the x-axis; PacBio read on the y-axis: ~1000 bp shift shows presence of insertion. Right panel: Inspection of the inserted sequence verified the presence of the L1 element (in blue) and the transduced sequence including the new polyA tail (in red; based on the consensus sequence created from all PacBio reads); the new polyadenylation signal is underlined. b Dotplot – with reference genome sequence on the x-axis and MinION read on the y-axis: ~1200 bp shift shows presence of an insertion. The inserted sequence verified both the presence of an L1 element (in blue) and additional transduced sequence including the new polyA tail (in red; based on the consensus sequence created from subset of MinION reads). c Alignment of the inserted L1 sequence to the ~6 kb long L1 consensus sequence shows that the integrated L1 is 5′-truncated (pairwise-alignment performed with BLAST)
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Fig4: Pacific Biosciences (a) and Oxford Nanopore MinION (b) long read verification of L1-mediated transduction insertions. aLeft panel: alignment dotplot – surrounding reference genome sequence for the human chr4:104210671-104214687 region shown on the x-axis; PacBio read on the y-axis: ~1000 bp shift shows presence of insertion. Right panel: Inspection of the inserted sequence verified the presence of the L1 element (in blue) and the transduced sequence including the new polyA tail (in red; based on the consensus sequence created from all PacBio reads); the new polyadenylation signal is underlined. b Dotplot – with reference genome sequence on the x-axis and MinION read on the y-axis: ~1200 bp shift shows presence of an insertion. The inserted sequence verified both the presence of an L1 element (in blue) and additional transduced sequence including the new polyA tail (in red; based on the consensus sequence created from subset of MinION reads). c Alignment of the inserted L1 sequence to the ~6 kb long L1 consensus sequence shows that the integrated L1 is 5′-truncated (pairwise-alignment performed with BLAST)

Mentions: Both short NGS reads and Sanger sequencing reads do not typically fully span the target locus, which complicates the characterization of transduction events. We reasoned that third generation long-read single molecule DNA sequencing technologies may help overcome this challenge, by fully recovering the complete sequence and structure of the combined insertion. Hence, we employed both Pacific Biosciences (PacBio) sequencing [40] (Fig. 4a and c) and Oxford Nanopore MinION sequencing (Fig. 4b and c) to obtain further insights into L1-mediated transductions.Fig. 4


Next-generation sequencing-based detection of germline L1-mediated transductions.

Tica J, Lee E, Untergasser A, Meiers S, Garfield DA, Gokcumen O, Furlong EE, Park PJ, Stütz AM, Korbel JO - BMC Genomics (2016)

Pacific Biosciences (a) and Oxford Nanopore MinION (b) long read verification of L1-mediated transduction insertions. aLeft panel: alignment dotplot – surrounding reference genome sequence for the human chr4:104210671-104214687 region shown on the x-axis; PacBio read on the y-axis: ~1000 bp shift shows presence of insertion. Right panel: Inspection of the inserted sequence verified the presence of the L1 element (in blue) and the transduced sequence including the new polyA tail (in red; based on the consensus sequence created from all PacBio reads); the new polyadenylation signal is underlined. b Dotplot – with reference genome sequence on the x-axis and MinION read on the y-axis: ~1200 bp shift shows presence of an insertion. The inserted sequence verified both the presence of an L1 element (in blue) and additional transduced sequence including the new polyA tail (in red; based on the consensus sequence created from subset of MinION reads). c Alignment of the inserted L1 sequence to the ~6 kb long L1 consensus sequence shows that the integrated L1 is 5′-truncated (pairwise-alignment performed with BLAST)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862182&req=5

Fig4: Pacific Biosciences (a) and Oxford Nanopore MinION (b) long read verification of L1-mediated transduction insertions. aLeft panel: alignment dotplot – surrounding reference genome sequence for the human chr4:104210671-104214687 region shown on the x-axis; PacBio read on the y-axis: ~1000 bp shift shows presence of insertion. Right panel: Inspection of the inserted sequence verified the presence of the L1 element (in blue) and the transduced sequence including the new polyA tail (in red; based on the consensus sequence created from all PacBio reads); the new polyadenylation signal is underlined. b Dotplot – with reference genome sequence on the x-axis and MinION read on the y-axis: ~1200 bp shift shows presence of an insertion. The inserted sequence verified both the presence of an L1 element (in blue) and additional transduced sequence including the new polyA tail (in red; based on the consensus sequence created from subset of MinION reads). c Alignment of the inserted L1 sequence to the ~6 kb long L1 consensus sequence shows that the integrated L1 is 5′-truncated (pairwise-alignment performed with BLAST)
Mentions: Both short NGS reads and Sanger sequencing reads do not typically fully span the target locus, which complicates the characterization of transduction events. We reasoned that third generation long-read single molecule DNA sequencing technologies may help overcome this challenge, by fully recovering the complete sequence and structure of the combined insertion. Hence, we employed both Pacific Biosciences (PacBio) sequencing [40] (Fig. 4a and c) and Oxford Nanopore MinION sequencing (Fig. 4b and c) to obtain further insights into L1-mediated transductions.Fig. 4

Bottom Line: We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome.We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species.By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Genome Biology Unit, 69117, Heidelberg, Germany.

ABSTRACT

Background: While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking.

Results: Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome. We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species.

Conclusions: By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.

No MeSH data available.


Related in: MedlinePlus