Targeted Nanopore Resequencing and Methylation Analysis of LINE-1 Retrotransposons - Inserm - Institut national de la santé et de la recherche médicale Accéder directement au contenu
Chapitre D'ouvrage Année : 2023

Targeted Nanopore Resequencing and Methylation Analysis of LINE-1 Retrotransposons

Résumé

Retrotransposition of LINE-1 (L1) elements represents a major source of insertional polymorphisms in mammals, and their mutagenic activity is restricted by silencing mechanisms, such as DNA methylation. Despite a very high level of sequence identity between copies, their internal sequence contains small nucleotide polymorphisms (SNPs) that can alter their activity. Such internal SNPs can also appear in different alleles of a given L1 locus. Given their repetitive nature and relatively long size, short-read sequencing approaches have limited access to L1 internal sequence or DNA methylation state. Here, we describe a targeted method to specifically sequence more than a hundred L1-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. Our protocol, modified from the nanopore Cas9 targeted sequencing (nCATS) strategy, provides a full and haplotype-resolved L1 sequence and DNA methylation levels. It introduces a streamlined and multiplex approach to synthesize guide RNAs and a quantitative PCR (qPCR)-based quality check during library preparation for cost-effective L1 sequencing. More generally, this method can be applied to any type of transposable elements and organisms.

Dates et versions

inserm-04492760 , version 1 (06-03-2024)

Licence

Copyright (Tous droits réservés)

Identifiants

Citer

Arpita Sarkar, Sophie Lanciano, Gael Cristofari. Targeted Nanopore Resequencing and Methylation Analysis of LINE-1 Retrotransposons. Miguel R. Branco; Alexandre de Mendoza Soler. Transposable Elements, 2607, Springer US, pp.173-198, 2023, Methods in Molecular Biology, 978-1-0716-2882-9. ⟨10.1007/978-1-0716-2883-6_10⟩. ⟨inserm-04492760⟩
4 Consultations
0 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More