Transposon insertion profiling by sequencing (TIPseq) for mapping LINE-1 insertions in the human genome

Abstract

Background Transposable elements make up a significant portion of the human genome. Accurately locating these mobile DNAs is vital to understand their role as a source of structural variation and somatic mutation. To this end, laboratories have developed strategies to selectively amplify or otherwise enrich transposable element insertion sites in genomic DNA. Results Here we describe a technique, Transposon Insertion Profiling by sequencing (TIPseq), to map Long INterspersed Element 1 (LINE-1, L1) retrotransposon insertions in the human genome. This method uses vectorette PCR to amplify species-specific L1 (L1PA1) insertion sites followed by paired-end Illumina sequencing. In addition to providing a step-by-step molecular biology protocol, we offer users a guide to our pipeline for data analysis, TIPseqHunter. Our recent studies in pancreatic and ovarian cancer demonstrate the ability of TIPseq to identify invariant (fixed), polymorphic (inherited variants), as well as somatically-acquired L1 insertions that distinguish cancer genomes from a patient’s constitutional make-up. Conclusions TIPseq provides an approach for amplifying evolutionarily young, active transposable element insertion sites from genomic DNA. Our rationale and variations on this protocol may be useful to those mapping L1 and other mobile elements in complex genomes. Electronic supplementary material The online version of this article (10.1186/s13100-019-0148-5) contains supplementary material, which is available to authorized users.

Human transposon insertion profiling by sequencing (TIPseq) to map LINE-1 insertions in single cells

“…It uses MDA for WGA, followed by a nested PCR using a primer specific to L1Hs ('AC', amplifying Ta and pre-Ta subfamilies) and degenerate primers [16]. Single-cell TIPseq is similar, but uses no nesting in its L1 amplification step; we use an L1 primer more specific to the Ta subfamily of L1Hs (ACA) [9] paired with a specific vectorette primer [42,47]. The latter requires additional steps to ligate sequences corresponding the vectorette primer to the DNA templates.…”

“…A pair of vectorette oligonucleotides (synthesized by IDT) corresponding to each restriction enzyme or T tail were annealed to form vectorette adaptors with the sticky end created. See sequences reported in [47]. Then the digested or repaired royalsocietypublishing.org/journal/rstb Phil.…”

“…LINE-1 insertions are frequent structural variants segregating in human populations, and many are not incorporated in the human reference genome assembly [34]. To understand genetic variation caused by these sequences and to find de novo insertions that distinguish cancer genomes from normal, many efforts have been made to profile LINE-1 insertions genome-wide using targeted or whole-genome sequencing [11,16,31,[35][36][37][38][39][40][41][42][43][44][45][46][47]. Our laboratories contributed an approach we termed transposon insertion profiling by sequencing (TIPseq) [31,41,42,47].…”

Integrated Mobile Element Scanning (ME-Scan) method for identifying multiple types of polymorphic mobile element insertions

“…Developed in the past few years, these methods included both PCR-based and probe-based enrichment strategies (Reviewed in [21]). PCR-based enrichment methods usually use a pair of primers to amplify the ME/genomic junction site: one primer that is specific to an ME of interest, and the 2nd primer that either binds to a generic linker sequence or to random genomic sequences [27][28][29][30][31][32][33]. The PCR-based methods have also been used lately with a multiplex modification [33,34].…”

“…Despite the advantage of low cost and high specificity, PCR-based methods usually focus on one specific type of ME [27][28][29][30][31][32][33]. To address this issue, we developed an integrated Mobile Element Scanning (ME-Scan) protocol building upon our previous ME-Scan protocols [28,29,39,40].…”