Apr 28, 2020

Detecting and Quantitating Low Fraction DNA Variants with Low-Depth Sequencing

BioRxiv : the Preprint Server for Biology
P. SongDavid Yu Zhang

Abstract

DNA sequence variants with low allele frequencies below 1% are difficult to detect and quantitate by sequencing, due to the intrinsic error of sequencing-by-synthesis (NGS). Unique molecular identifier barcodes can in principle help NGS detect mutations down to 0.1% variant allele frequency (VAF), but require extremely high sequencing depths of over 25,000x, rendering high sensitivity mutation detection out of reach for most research and clinical samples. Here, we present the multiplex blocker displacement amplification (mBDA) method to selectively enrich DNA variants by an average of 300-fold in highly multiplexed NGS settings. On a 80-plex human single nucleotide polymorphism panel, mBDA achieves a 0.019% VAF limit of detection for single nucleotide variants, using only 250x sequencing depth, and detects human cell line contamination down to 0.07%. Using this technology, we constructed a 16-plex melanoma NGS panel covering 145 actionable mutations across 9 genes, and applied it to 19 fresh/frozen tumor biopsy tissue samples with high tumor fractions. We found low VAF mutations (0.2% to 5%) in 37% of the samples (7/19, 95% confidence interval 19%-58%). These results suggest that tumor heterogeneity could be significantly more ...Continue Reading

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Mentioned in this Paper

Metabolic Process, Cellular
DNA Repair
Transcription, Genetic
Open Label Study
Virus Replication
Molecular_function
Prader-Willi Syndrome
Partial Wave Spectroscopic Microscopy
Mitochondrial Membranes
SPT6 Protein

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