Abstract
Genotyping of cell-free DNA (cfDNA) in plasma samples has the potential to allow for a noninvasive assessment of tumor biology, avoiding the inherent shortcomings of tissue biopsy. Next generation sequencing (NGS), a leading technology for liquid biopsy analysis, continues to be hurdled with several major issues with cfDNA samples, including low cfDNA concentration and high fragmentation. In this study, by employing Ion Torrent PGM semiconductor technology, we performed a comparison between two multi-biomarker amplicon-based NGS panels characterized by a substantial difference in average amplicon length. In course of the analysis of the peripheral blood from 13 diagnostic non-small cell lung cancer patients, equivalence of two panels, in terms of overall diagnostic sensitivity and specificity was shown. A pairwise comparison of the allele frequencies for the same somatic variants obtained from the pairs of panel-specific amplicons, demonstrated an identical analytical sensitivity in range of 140 to 170 bp amplicons in size. Further regression analysis between amplicon length and its coverage, illustrated that NGS sequencing of plasma cfDNA equally tolerates amplicons with lengths in the range of 120 to 170 bp. To increase the sensitivity of mutation detection in cfDNA, we performed a computational analysis of the features associated with genome-wide nucleosome maps, evident from the data on the prevalence of cfDNA fragments of certain sizes and their fragmentation patterns. By leveraging the support vector machine-based machine learning approach, we showed that a combination of nucleosome map associated features with GC content, results in the increased accuracy of prediction of high inter-sample sequencing coverage variation (areas under the receiver operating curve: 0.75, 95% CI: 0.750–0.752 vs. 0.65, 95% CI: 0.63–0.67). Thus, nucleosome-guided fragmentation should be utilized as a guide to design amplicon-based NGS panels for the genotyping of cfDNA samples.
Highlights
In an approach known as “liquid biopsy,” cell-free DNA which circulates in the plasma may be used for a diagnostic detection of tumor-specific mutations (Dawson et al, 2013; Pupilli et al, 2013; Xi et al, 2016)
We investigated the effect of the amplicon length on the diagnostic and analytical sensitivity of mutation detection, using two amplicon-based Next generation sequencing (NGS) panels with diverse amplicon lengths
Fourteen cell-free DNA (cfDNA) samples collected from patients with non-small cell lung cancer (NSCLC), were analyzed using the screening panels ITCHP2 and AODCP
Summary
In an approach known as “liquid biopsy,” cell-free DNA (cfDNA) which circulates in the plasma may be used for a diagnostic detection of tumor-specific mutations (Dawson et al, 2013; Pupilli et al, 2013; Xi et al, 2016). PCR-based and hybridizationbased cfDNA profiling techniques are developed to detect particular DNA variants, which most commonly underlie one or another previously described pathophysiological process These and other variant-specific techniques are not suitable for the exploratory analysis of cfDNA, which is necessary for acquisition of knowledge concerning non-conventional, emerging resistance pathways, for co-detection of the mismatch repair phenotype, and for off-label prescribing of anticancer medications commonly required for personalized treatment of metastatic tumors (Tafe et al, 2015; Wei et al, 2016; Zehir et al, 2017). These limitations are readily surmounted by an advent of sequencing-based technologies, including whole exome sequencing or, more applicable to cfDNA analysis, ampliconbased panels, which are limited to their target genes, but are still exploration-permissive
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