Program > Browse abstracts by author > Voshall Adam

The human genome is mosaic in tissues and shaped by a myriad of mutational processes. Our group studies its only active autonomous retrotransposon LINE-1(L1), which generates novel insertions through a “copy and paste” mechanism of RNA reverse transcription. These de novo insertions can interrupt genes or alter their expression, generate chromosomal rearrangements, or generally contribute to DNA damage and genome instability. Somatic L1 activity is best studied in cancers, with the L1 protein ORF1p present across a variety of cancer types, while recent work has demonstrated somatic activity in normal tissue including the brain and the colon. 17% of the human genome is made up of L1 sequence, yet only about 100 copies are intact and capable of retrotransposition in an individual. Many active L1 loci (“hot” L1) are genetic variants in human populations and thus differ from individual to individual. In a given tissue or cell type, a subset of these may be expressed. Defining active L1 loci has been challenging due in-part to the limited abilities of short read sequencing to accurately map to and cover the full length of L1 insertions, and the difficulties in then combining that sequence data with locus specific methylation data. Here we present a Cas9 targeted long read approach using Nanopore which allows us to sequence the entirety of genomic L1 insertions with their flanking regions and capture the methylation status of each locus directly from the genomic DNA. We combine this protocol with a novel in silico pipeline to analyze targeted Nanopore reads from repeat elements and can integrate it with existing L1 insertion detection tools. This technique will enable the field to continue to refine our understanding of “hot” L1 activity.