RLB-MALBAC: a rapid, non-transposase method for synthesizing unbiased sequencing libraries from low amount of genomic DNA for Nanopore sequencing

Abstract Background Successful sequencing of genomes on Oxford Nanopore Technologies (ONT) sequencing platforms is dependant on obtaining sufficient amounts of high-quality genomic DNA. ONT’s Rapid PCR kit (SQK-RPB114.24) kit is specifically designed to address input template limitations, whereby low amounts of target DNA (1–5 ng) is pre-amplified to > 100–200 ng using PCR. The success of this workflow is however dependent on the quality of input DNA, which as per the SQK-RPB114.24 kit’s instructions, should be of length > 4 kb for an efficient tagmentation—a step pre-cursor to PCR amplification. Obtaining high quality, intact genomic DNA from clinical samples or viral genomes, can sometimes be challenging and this method seeks to address this limitation. Methods In this study, a novel non-tagmentation based method is presented to prepare sequencing libraries from low amounts of genomic DNA (< 5–100 ng). Our method, based on a quasilinear amplification method, the Multiple Annealing and Looping Based Amplification Cycles chemistry (MALBAC), randomly introduces a 21-nucleotide, PCR anchor (“RLB tag”), across the target. This method, henceforth referred to as RLB-MALBAC, results in the generation of RLB-tagged genomic libraries which are then compatible with the PCR dependent RLB-BC01-24 series barcodes of the SQK-RPB114.24 kit. Results The RLB-MALBAC method was used to sequence genomes from Escherichia coli bacteriophage lambda (48.5 kb) virus as well as clinically relevant bacteria (2–5 Mb genome range). To demonstrate one potential application of RLB-MALBAC, we tested the generated contigs for the presence of antimicrobial resistance (AMR) genes of the β-lactamase class. Using the Comprehensive Antibiotic Resistance Database (CARD) prediction tool, we found a 100% concordance between the β-lactamase genes predicted by RLB-MALBAC and the reference sequences (Illumina + Nanopore hybrid assemblies) for ATCC bacterial isolates. We also predicted resistance phenotypes from clinical bacterial isolates confirmed to carry β-lactamases. Discussion As the SQK-RPB114.24 kit is PCR dependent, a short read-length (2-3 kb) is expected using the RLB-MALBAC method, which is a major limitation of this workflow. The versatility and simplicity of the RLB-MALBAC method should find broad application in other areas of microbiology, for e.g., plasmid, viral and single cell genomics, where there may be need to amplify from limited amounts of genomic material.

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