Beyond detection — adding context to viral integrations patterns
Integrations of viral DNA into host genomes can alter gene expression, predispose disease-causing mutations or rearrangements, and even shape how the host genome evolves.
Analyzing insertions within the context of the host integration sequence is important to understanding the role of insertions in plant biology, immune responses, cancer and more.
To that end, enriching long DNA fragments containing the low-abundance viral target makes it easier to map and resolve insertion patterns. However, methods based on long-range PCR or CRISPR-Cas to enrich such molecules are laborious to set up, challenging to design, and cannot easily access long and unknow regions. But not Xdrop™.
Using Xdrop™, we enriched long fragments of a ~30 kb-long region of DNA isolated from HeLa cells that contained segments of the HPV18 genome.
Briefly, we sorted out single molecules of interest based on the presence of a specific short sequence (Detection Sequence in the figure) in the viral genome and subjected each to multiple displacement amplification to create an unbiased 500 to 1000-fold enrichment, as determined by sequencing on 3 platforms.
We then prepared the enriched long DNA fragments for short-read sequencing on the MiSeq and long-read sequencing on the PacBio and Oxford Nanopore platforms.
Mapped to the HPV18 genome, the resulting reads showed consistent hallmarks across all platforms: a central part of the viral genome was absent in inserts and abrupt drops in coverage aligned with fusion points where reads transitioned from viral to human sequences. Download the Application Note to see our mapping to chromosome 8.
Because they spanned the insertion fusion points, our enriched long DNA fragments revealed 3 different integration variants of HPV18 into chromosome 8. The variants differed in insertion length and in the fusion point at one end. All fusion points were validated by PCR and Sanger sequencing.
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