Mobile element scanning (ME-Scan) by targeted high-throughput sequencing

Abstract

BackgroundMobile elements (MEs) are diverse, common and dynamic inhabitants of nearly all genomes. ME transposition generates a steady stream of polymorphic genetic markers, deleterious and adaptive mutations, and substrates for further genomic rearrangements. Research on the impacts, population dynamics, and evolution of MEs is constrained by the difficulty of ascertaining rare polymorphic ME insertions that occur against a large background of pre-existing fixed elements and then genotyping them in many individuals.ResultsHere we present a novel method for identifying nearly all insertions of a ME subfamily in the whole genomes of multiple individuals and simultaneously genotyping (for presence or absence) those insertions that are variable in the population. We use ME-specific primers to construct DNA libraries that contain the junctions of all ME insertions of the subfamily, with their flanking genomic sequences, from many individuals. Individual-specific "index" sequences are designed into the oligonucleotide adapters used to construct the individual libraries. These libraries are then pooled and sequenced using a ME-specific sequencing primer. Mobile element insertion loci of the target subfamily are uniquely identified by their junction sequence, and all insertion junctions are linked to their individual libraries by the corresponding index sequence. To test this method's feasibility, we apply it to the human AluYb8 and AluYb9 subfamilies. In four individuals, we identified a total of 2,758 AluYb8 and AluYb9 insertions, including nearly all those that are present in the reference genome, as well as 487 that are not. Index counts show the sequenced products from each sample reflect the intended proportions to within 1%. At a sequencing depth of 355,000 paired reads per sample, the sensitivity and specificity of ME-Scan are both approximately 95%.ConclusionsMobile Element Scanning (ME-Scan) is an efficient method for quickly genotyping mobile element insertions with very high sensitivity and specificity. In light of recent improvements to high-throughput sequencing technology, it should be possible to employ ME-Scan to genotype insertions of almost any mobile element family in many individuals from any species.

Reading TE leaves: New approaches to the identification of transposable element insertions

“…For example, by utilizing Illumina sequencing technology, Ewing and Kazazian (2010b) and Witherspoon et al (2010) were able to scan entire genomes of multiple individuals to identify polymorphisms. However, as is often the case, the cost of so many reads comes in the form of reduced read length and both studies are somewhat limited in their ability to query the human genome reference, especially in highly repetitive regions.…”

“…Similarly, Witherspoon et al (2010) utilized Illumina technology, but with a different method that targets the genomic sequence junctions of Alu elements. Subsequent steps enriched for Alu-containing PCR amplicons and the resulting libraries were sequenced using a paired-end protocol.…”

“…However, the studies discussed herein identified numerous insertions with the potential to be useful in this area. For example, Ewing and Kazazian (2010b) identified over 300 nonreference L1 insertions while Witherspoon et al (2010) simultaneously identified and mapped nearly 500 novel polymorphic Alu insertions in four individuals. Additionally, Beck et al (2010) identified three L1 insertions apparently restricted to persons of African origin.…”

“…Whereas only 10 yr ago we were able to generate at best a few hundred thousand bases per day using chain termination sequencing methods, we can now generate gigabases of data in a single run of a 454 (Roche) or Illumina machine. Five recent papers report multiple methods based on second-generation sequencing techniques as well as hybridization arrays to rapidly and relatively inexpensively characterize genome-wide TE insertion patterns (Beck et al 2010;Ewing and Kazazian 2010b;Huang et al 2010;Iskow et al 2010;Witherspoon et al 2010) and identify a plethora of human TE polymorphisms. With these novel methods, the identification of markers for a vast array of applications can be specifically targeted.…”

Retrotransposons and Complex Diseases: Is it Time for a Retrotransposon- Based “Omics” Profiling approach to Elucidate the Origins of Pathogenesis?

“…GID data resulting from whole-genome sequencing (WGS) coupled with RNA sequencing (RNA-Seq) and Proteomic analyses will be exploitable to decipher both the genomic architecture and the pathogenic variants. To this direction, it would be of great help the existing approaches for identification of the LINE-1 and Alu mobilome designed and executed until now [17][18][19]. Nevertheless, the development of novel experimental methodologies, which can support and supplement the existing ones is necessary, in order to determine the whole human mobilome.…”