Increased detection of circulating tumor DNA by short fragment enrichment

Abstract

Background: Circulating cell-free DNA (cfDNA) detection for non-invasive diagnosis requires higher sensitivity and accuracy due to the low circulating tumor DNA (ctDNA) content. Many methods have been developed to improve detection of ctDNA, including ultra-deep sequencing or enrichment of shorter cfDNA fragments, such as those in the range of 90-150 bp.Methods: Here, we developed a method for single-stranded DNA (ssDNA) library preparation with a large proportion of magnetic beads to enrich the shorter cfDNA fragments. We aimed to determine if this could increase the ctDNA content and thus improve the sensitivity of ctDNA detection by testing the method in blood samples from patients with advanced cancers (non-small cell lung cancers, esophageal squamous cell carcinoma, cholangiocarcinoma, colorectal cancer and liver cancer).Results: This method was able to obtain shorter cfDNA both in commercial cfDNA references and real world clinical cfDNA samples. Plasmid simulation experiments showed that using a large proportion of magnetic beads to construct the library could obtain more ctDNA derived from shorter-fragment plasmids, which could significantly improve the detection of ctDNA especially in the low-variant allele frequency sample. In real-world clinical samples, this method may be able to increase the opportunity to obtain alteration reads from short fragments, which was important to low frequency detection. Conclusions:The ssDNA library preparation with large proportion of magnetic beads could increase the opportunity to obtain alteration reads from short fragments, which is crucial for low variant allele frequency detection.

Transcending Blood—Opportunities for Alternate Liquid Biopsies in Oncology

“…The release process generally results in a distinctive, ladder-like fragmentation pattern that reflects cleavage between nucleosomes, with the nucleosomes protecting the DNA from further degradation [ 75 ]. The size profile of cirDNA gives an insight into its origin: cirDNA fragments of ~167 base pairs are associated with apoptosis where they arise from caspase-dependent cleavage [ 53 ]; longer cirDNA fragments may originate from necrotic cells or extracellular vesicles released actively by live cells [ 73 , 74 ]; tumour derived cirDNA is enriched in smaller, mono-nucleosomal fragments of 90–150 base pairs [ 76 , 77 , 78 ].…”

Tracking the evolution of untreated high‐intermediate/high‐risk diffuse large B‐cell lymphoma by circulating tumour DNA

“…To overcome this shortcoming, we employed a DLBCL‐focussed sequencing panel targeting recurrent amplification, copy number gain, insertions/deletion mutations, and breakpoints involving genes that participate in canonical fusions using a next‐generation sequencing (NGS)‐based methodology called circulating single‐molecule amplification and re‐sequencing technology (cSMART). ctDNA is usually prone to fragmentation resulting in reducing the sensitivity of polymerase chain reaction (PCR)‐based methods due to incapable binding of one primer 6 . cSMART has a high detection sensitivity and specificity by circularization of tagged DNA molecules and using target‐specific back‐to‐back primer pair tools to make the sequences inversely amplified and thus retaining the molecule barcodes and original length information 7 .…”

Comparative Panel Sequencing of DNA Variants in cf-, ev- and tumorDNA for Pancreatic Ductal Adenocarcinoma Patients

“…These data indicate that the parameters ctDNA or tumor EV fraction must be considered when planning clinical studies or implementing liquid biopsy analysis in the clinical routine. To this end, the tumor-derived cfDNA fraction may be quantified or enriched by focusing on smaller fragmented DNA sizes [ 62 ]. Similar considerations apply to tumor EVs.…”