Fig. 2.

Concatenation of short amplicons by Stochastic Amplicon Ligation (SAL). a The number of Nanopore Sequencing reads per hour observed for two different lengths of DNA using SQK-LSK109 sample prep chemistry on R9.4.1 flow cells. Here, we performed Nanopore Sequencing on the NA18562 human genomic DNA (gDNA) physically sheared (Covaris g-tube) to an average of 265 base pairs (bp) or 3800 bp (3.8 kb). The effective throughput of the longer gDNA library was roughly 10-fold higher than the shorter gDNA library. b Concatenation of DNA amplicons through SAL. Amplicons are adapted so that their 5 ^′ and 3 ^′ ends possess Type IIS restriction enzyme sites. Through a series of restriction and ligation reactions, >10 amplicons are assembled into concatemers. Compared to a naive method based on blunt end ligation, SAL enriches longer concatemers by maintaining low concentrations of amplicon monomers during each cycle of the reaction. Additionally, SAL significantly increases the on-target rate of the final Nanopore Sequencing libraries by excluding undesired dsDNA molecules from being incorporated into the concatemer. c Capillary electrophoresis analysis of a 220 nt amplicon and its SAL concatemer products. d Nanopore Sequencing read lengths of concatemers for a 7-plex SAL reaction from amplicons with a mean length of 340 nt. In addition to the increased throughput of Nanopore Sequencing for the concatemer due to the longer DNA lengths, we also observed a significant increase in sequencing quality for the concatemer vs. the original amplicon. e Increased Nanopore Sequencing throughput from SAL improves the limit of detection of somatic mutations when paired normal samples are available. The top traces show the variant read fraction (VRF) of Nanopore Sequencing reads at each location that corresponds to the highest frequency single-base changes (substitution, insertion deletion). The bottom traces show the relative excess of the top variant at each position for the 5% VAF samples compared to a 0% VAF sample; see Additional file 1: Fig. S7 for amplicon Nanopore Sequencing traces for 0% VAF samples. The two SNP alleles specific to NA18562 were more prominently called in the SAL concatemer Nanopore Sequencing results. The input DNA for each run was either 50 ng of a 95%:5% mixture of the NA18537 and NA18562 cell line human genomic DNA (gDNA), or 50 ng NA18537. Note: The results in panels a, d and e are specifically using SQK-LSK109 sample prep chemistry on R9.4.1 flow cells using MinKNOW 19.12.5 for basecalling