Abstract
BackgroundTransposable elements (TEs) are mobile genetic sequences that randomly propagate within their host’s genome. This mobility has the potential to affect gene transcription and cause disease. However, TEs are technically challenging to identify, which complicates efforts to assess the impact of TE insertions on disease. Here we present a targeted sequencing protocol and computational pipeline to identify polymorphic and novel TE insertions using next-generation sequencing: TE-NGS. The method simultaneously targets the three subfamilies that are responsible for the majority of recent TE activity (L1HS, AluYa5/8, and AluYb8/9) thereby obviating the need for multiple experiments and reducing the amount of input material required.ResultsHere we describe the laboratory protocol and detection algorithm, and a benchmark experiment for the reference genome NA12878. We demonstrate a substantial enrichment for on-target fragments, and high sensitivity and precision to both reference and NA12878-specific insertions. We report 17 previously unreported loci for this individual which are supported by orthogonal long-read evidence, and we identify 1470 polymorphic and novel TEs in 12 additional samples that were previously undocumented in databases of insertion polymorphisms.ConclusionsWe anticipate that future applications of TE-NGS alongside exome sequencing of patients with sporadic disease will reduce the number of unresolved cases, and improve estimates of the contribution of TEs to human genetic disease.
Highlights
Transposable elements (TEs) are mobile genetic sequences that randomly propagate within their host’s genome
We first prepare genomic libraries using standard next generation sequencing (NGS) procedures followed by polymerase chain reaction (PCR) enrichment of library fragments containing TE insertions and their flanking regions
Exponential amplification is achieved by multiplex PCR of the two TE-target primers each in conjunction with the Illumina Universal (P5) primer that will anneal to the complimentary i5 adapter present on all library molecules
Summary
Transposable elements (TEs) are mobile genetic sequences that randomly propagate within their host’s genome. This mobility has the potential to affect gene transcription and cause disease. Recent large-scale sequencing efforts of individuals with rare Mendelian disorders indicate that in approximately 20–40% of cases a causal variant can be identified (eg, [1,2,3,4]). To date, these efforts have focused mainly on small variants in exonic regions.
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