ABSTRACT
Human papillomavirus (HPV) integration has been implicated in transforming HPV infection into cancer, but its genomic consequences have been difficult to study using short-read technologies. To resolve the dysregulation associated with HPV integration, we performed long-read sequencing on 63 cervical cancer genomes. We identified six categories of integration events based on HPV-human genomic structures. Of all HPV integrants, defined as two HPV-human breakpoints bridged by an HPV sequence, 24% contained variable copies of HPV between the breakpoints, a phenomenon we termed heterologous integration. Analysis of DNA methylation within and in proximity to the HPV genome at individual integration events revealed relationships between methylation status of the integrant and its orientation and structure. Dysregulation of the human epigenome and neighboring gene expression in cis with the HPV-integrated allele was observed over megabase-ranges of the genome. By elucidating the structural, epigenetic, and allele-specific impacts of HPV integration, we provide insight into the role of integrated HPV in cervical cancer.
ABSTRACT
Single-cell Strand-seq generates directional genomic information to study DNA repair, assemble genomes, and map structural variation onto chromosome-length haplotypes. We report a nanoliter-volume, one-pot (OP) Strand-seq library preparation protocol in which reagents are added cumulatively, DNA purification steps are avoided, and enzymes are inactivated with a thermolabile protease. OP-Strand-seq libraries capture 10%-25% of the genome from a single-cell with reduced costs and increased throughput.
Subject(s)
Genomics , Genomics/methods , HaplotypesABSTRACT
Mammalian genomes encode over a hundred different helicases, many of which are implicated in the repair of DNA lesions by acting on DNA structures arising during DNA replication, recombination or transcription. Defining the in vivo substrates of such DNA helicases is a major challenge given the large number of helicases in the genome, the breadth of potential substrates in the genome and the degree of genetic pleiotropy among DNA helicases in resolving diverse substrates. Helicases such as WRN, BLM and RECQL5 are implicated in the resolution of error-free recombination events known as sister chromatid exchange events (SCEs). Single cell Strand-seq can be used to map the genomic location of individual SCEs at a resolution that exceeds that of classical cytogenetic techniques by several orders of magnitude. By mapping the genomic locations of SCEs in the absence of different helicases, it should in principle be possible to infer the substrate specificity of specific helicases. Here we describe how the genome can be interrogated for such DNA repair events using single-cell template strand sequencing (Strand-seq) and bioinformatic tools. SCEs and copy-number alterations were mapped to genomic locations at kilobase resolution in haploid KBM7 cells. Strategies, possibilities, and limitations of Strand-seq to study helicase function are illustrated using these cells before and after CRISPR/Cas9 knock out of WRN, BLM and/or RECQL5.
Subject(s)
DNA Replication , Sister Chromatid Exchange , Animals , DNA/chemistry , DNA/genetics , DNA Repair/genetics , DNA Replication/genetics , Genome , Mammals , Sister Chromatid Exchange/geneticsABSTRACT
Maintenance of genome stability is essential to prevent the accumulation of DNA mutations that can initiate oncogenesis and facilitate tumor progression. Studies of DNA repair genes have revealed a highly dynamic and redundant network of genes and proteins responsible for maintaining genome stability. Cancer cells are often deficient in DNA repair, and the resulting genome instability decreases their fitness but also allows for more rapid evolution under selective pressure. Of particular interest for genome stability are the RecQ class of helicases. Five genes in this class, RECQL1, BLM, WRN, RECQL4, and RECQL5, are unique to mammals, as simpler eukaryotes and bacteria appear to have only one homolog, RecQ. The precise role of each of the five mammalian RecQ helicases remains to be determined. Whereas loss of function mutations of BLM, WRN, and RECQL4 in humans are associated with specific diseases, RECQL1 and RECQL5 have not yet been associated with specific disorders. Mice deficient in Recql5 are more likely to develop cancer, and human cells deficient in RECQL5 display chromosomal instability and elevated sister chromatid exchange events, similar to cells deficient in any of the other RecQ helicases. Recent studies support the hypothesis that RECQL5 can resolve intermediate DNA repair structures resulting from the collision of DNA transcription and replication machinery. In this review, we aim to summarize current knowledge regarding RECQL5 in the context of DNA repair, replication, and transcription to help uncover the role of RECQL5 in the maintenance of genome stability.