Duplex Proximity Sequencing (Pro-Seq): A method to improve DNA sequencing accuracy without the cost of molecular barcoding redundancy

Abstract

A challenge in the clinical adoption of cell-free DNA (cfDNA) liquid biopsies for cancer care is their high cost compared to potential reimbursement. The most common approach used in liquid biopsies to achieve high specificity detection of circulating tumor DNA (ctDNA) among a large background of normal cfDNA is to attach molecular barcodes to each DNA template, amplify it, and then sequence it many times to reach a low-error consensus. In applications where the highest possible specificity is required, error rate can be lowered further by independently detecting the sequences of both strands of the starting cfDNA. While effective in error reduction, the additional sequencing redundancy required by such barcoding methods can increase the cost of sequencing up to 100-fold over standard next-generation sequencing (NGS) of equivalent depth. We present a novel library construction and analysis method for NGS that achieves comparable performance to the best barcoding methods, but without the increase in sequencing and subsequent sequencing cost. Named Proximity-Sequencing (Pro-Seq), the method merges multiple copies of each template into a single sequencing read by physically linking the molecular copies so they seed a single sequencing cluster. Since multiple DNA copies of the same template are compared for consensus within the same cluster, sequencing accuracy is improved without the use of redundant reads. Additionally, it is possible to represent both senses of the starting duplex in a single cluster. The resulting workflow is simple, and can be completed by a single technician in a work day with minimal hands on time. Using both cfDNA and cell line DNA, we report the average per-mutation detection threshold and per-base analytical specificity to be 0.003% and >99.9997% respectively, demonstrating that Pro-Seq is among the highest performing liquid biopsy technologies in terms of both sensitivity and specificity, but with greatly reduced sequencing costs compared to existing methods of comparable accuracy.

Systematic comparison of somatic variant calling performance among different sequencing depth and mutation frequency

“…Hence, other methods providing high performance for detecting low frequency mutations are needed to be applied. Many error correction methods on WES have been developed and commonly used nowadays [27][28][29][30][31][32][33] , the unique molecular identifiers (UMIs) based error correction strategy is very useful in the above context. UMIs can tag sequences to help track molecules and remove errors in amplification and sequencing [34][35][36][37][38] , Michael et al developed a method called "duplex sequencing" which tagged the sequencing adapter by a degenerated 12 randomized base (Duplex Tag) and 4 base length fixed sequence 27 .…”

Next-Generation Sequencing in High-Sensitive Detection of Mutations in Tumors

“…For instance, duplex proximity sequencing physically links copies of every template DNA strand so that both the duplex strands are sequenced as a single cluster during sequencing, and the mutant detected in the cluster can be traced back to both DNA strands (þ and À) without the need for duplex sequencing with UIDs. 62 Similarly, in another UID-free method, post-NGS sequencing, the reads that have low-level mutations are physically and selectively isolated and amplified and the sequence is confirmed by either NGS or Sanger sequencing, thus avoiding the need to sequence the wild-type sequence background. 63 Furthermore, a recent study has demonstrated the use of technical replicates from the level of library preparation as a solution to accurately detect low-level mutations without using UID tagging.…”

Detection of RAS mutations in circulating tumor DNA: a new weapon in an old war against colorectal cancer. A systematic review of literature and meta-analysis

“…Modern NGS systems produce errors at a per-base rate of 10 −2 to 10 −3 , but clinically relevant mutations have been revealed to be at, or below, that level making many true variants undetectable. 47 In recent years error correction strategies have been developed and today NGS and liquid biopsy have a great potential in a gene’s mutational status detection and impact positively on diagnostics and cancer management. Recently, ctDNA liquid biopsies have been adopted for several personalized medicine applications, including guiding treatment selection during monitoring, minimum residual disease detection, and even screening (ClinicalTrials.gov NCT02808884; NCT02889978).…”