Retrotransposon Capture Sequencing (RC-Seq): A Targeted, High-Throughput Approach to Resolve Somatic L1 Retrotransposition in Humans

Abstract

Mobile genetic elements (MGEs) are of critical importance in genomics and developmental biology. Polymorphic and somatic MGE insertions have the potential to impact the phenotype of an individual, depending on their genomic locations and functional consequences. However, the identification of polymorphic and somatic insertions among the plethora of copies residing in the genome presents a formidable technical challenge. Whole genome sequencing has the potential to address this problem; however, its efficacy depends on the abundance of cells carrying the new insertion. Robust detection of somatic insertions present in only a subset of cells within a given sample can also be prohibitively expensive due to a requirement for high sequencing depth. Here, we describe retrotransposon capture sequencing (RC-seq), a sequence capture approach in which Illumina libraries are enriched for fragments containing the 5' and 3' termini of specific MGEs. RC-seq allows the detection of known polymorphic insertions present in an individual, as well as the identification of rare or private germline insertions not previously described. Furthermore, RC-seq can be used to detect and characterize somatic insertions, providing a valuable tool to elucidate the extent and characteristics of MGE activity in healthy tissues and in various disease states.

Human transposon insertion profiling: Analysis, visualization and identification of somatic LINE-1 insertions in ovarian cancer

“…The reference genome assembly captures fixed alleles and high-frequency alleles, but does not encompass many common variants (17). Targeted methods for recovering LINE-1 insertions for next-generation sequencing are new technologies, and include approaches developed by Rahbari and Badge (38), Devine and co-workers (15), Faulkner and coworkers (19,39,40), Kazazian and co-workers (16,41), Deininger and co-workers (42), and Gage and co-workers (43), as well as our own approaches. Collectively, their application has reinforced that LINE-1 is an important source of structural variation in humans and contributed to a growing database of LINE-1 insertion polymorphisms (44).…”

LINE-1 retrotransposition impacts the genome of human pre-implantation embryos and extraembryonic tissues

“…Briefly, we isolated single cells from the ICM of hEMBs that reach the blastocyst stage (Figure 1a, 3b), amplified their genomes using Multiple Displacement Amplification (MDA, (Spits et al, 2006)), and analyzed the presence of new insertions using two different Next Generation DNA sequencing protocols: Retrotransposon Capture Sequencing (RC-Seq, (Sanchez-Luque et al, 2016)) and Amplification Typing of L1 Active Subfamilies Sequencing (ATLAS -Seq, (Philippe et al, 2016)) (Figure 3a and Text File S1). As validation of de novo LINE-1 insertions in MDA-amplified DNAs is extremely challenging (Evrony et al, 2012), we reasoned that combining two independent LINE-1 profiling methods on the same sample could help us to limit method -specific artifacts resulting from library preparation, sequencing or bioinformatic analyses (Figure 3a and Text File S1).…”

“…Next, we applied RC-seq to H9-hESCs at passages p58 and p69 (Sanchez-Luque et al, 2016;Upton et al, 2015), using high molecular weight (HMW) DNAs (Figure 5d). Upon bioinformatic analyses to remove annotated and polymorphic L1Hs insertions (see Text File S2 for additional details on validation of insertions in hESCs), we validated and characterized a de novo LINE-1 retrotransposition event (H9-16-723) (Figure 5d-g).…”

SOX‐11 regulates LINE‐1 retrotransposon activity during neuronal differentiation

“…Differential LINE‐1 activity in single neurons is believed to play a key role in brain mosaicism, with major effects on neuronal circuits and/or in determining neuron fate. However, in our study, the identity of the LINE‐1 elements retrotransposed and their localisation in the genome remains to be established, a goal that could be achieved by the newly developed technique Single‐cell retrotransposon capture sequencing . This technology has shed light on LINE‐1 mosaicism at genomic loci expressed in hippocampal neurons: in a report by Upton et al .…”