ConcatSeq: A method for increasing throughput of single molecule sequencing by concatenating short DNA fragments

Single molecule sequencing (SMS) platforms enable base sequences to be read directly from individual strands of DNA in real-time. Though capable of long read lengths, SMS platforms currently suffer from low throughput compared to competing short-read sequencing platforms. Here, we present a novel strategy for sequencing library preparation, dubbed ConcatSeq, which increases the throughput of SMS platforms by generating long concatenated templates from pools of short DNA molecules. We show adaptation of this technique to two target enrichment workflows, commonly used for oncology applications, and demonstrate feasibility using PacBio single molecule real-time (SMRT) Technology. We demonstrate that our approach increases the sequencing throughput of the PacBio RSII platform by more than five-fold and that allele frequencies of known single nucleotide variants in oncology targets are called with high accuracy and sensitivity. ConcatSeq therefore provides a powerful and versatile new sample preparation tool for long-read sequencing technologies.