10.5061/DRYAD.7PJ51V0
Hellwig, Sabine
Nix, David A.
University of Utah
Gligorich, Keith M.
University of Utah
O'Shea, John M.
Thomas, Alun
University of Utah
Fuertes, Carrie L.
University of Utah
Bhetariya, Preetida J.
University of Utah
Marth, Gabor T.
University of Utah
Bronner, Mary P.
University of Utah
Underhill, Hunter R.
University of Utah
O’Shea, John M.
University of Utah
Data from: Automated size selection for short cell-free DNA fragments
enriches for circulating tumor DNA and improves error correction during
next generation sequencing
Dryad
dataset
2019
Next-generation sequencing
cell-free DNA
Melanoma
digital droplet PCR
pancreatic ductal adenocarcinoma
colorectal adenocarcinoma
2019-08-03T00:00:00Z
2019-08-03T00:00:00Z
en
https://doi.org/10.1371/journal.pone.0197333
367773039010 bytes
1
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
Circulating tumor-derived cell-free DNA (ctDNA) enables non-invasive
diagnosis, monitoring, and treatment susceptibility testing in human
cancers. However, accurate detection of variant alleles, particularly
during untargeted searches, remains a principal obstacle to widespread
application of cell-free DNA in clinical oncology. In this study,
isolation of short cell-free DNA fragments is shown to enrich for tumor
variants and improve correction of PCR- and sequencing-associated errors.
Subfractions of the mononucleosome of circulating cell-free DNA (ccfDNA)
were isolated from patients with melanoma, pancreatic ductal
adenocarcinoma, and colorectal adenocarcinoma using a
high-throughput-capable automated gel-extraction platform. Using a
128-gene (128 kb) custom next-generation sequencing panel, variant alleles
were on average 2-fold enriched in the short fraction (median insert size:
~142 bp) compared to the original ccfDNA sample, while 0.7-fold reduced in
the fraction corresponding to the principal peak of the mononucleosome
(median insert size: ~167 bp). Size-selected short fractions compared to
the original ccfDNA yielded significantly larger family sizes (i.e., PCR
duplicates) during in silico consensus sequence interpretation via unique
molecular identifiers. Increments in family size were associated with a
progressive reduction of PCR and sequencing errors. Although consensus
read depth also decreased at larger family sizes, the variant allele
frequency in the short ccfDNA fraction remained consistent, while variant
detection in the original ccfDNA was commonly lost at family sizes
necessary to minimize errors. These collective findings support the
automated extraction of short ccfDNA fragments to enrich for ctDNA while
concomitantly reducing false positives through in silico error correction.
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