Towards a rapid-turnaround low-depth unbiased metagenomics sequencing workflow on the Illumina platforms

Abstract

AbstractUnbiased metagenomic sequencing is conceptually well-suited for first-line infectious disease diagnostics because it does not require guesswork on the causative organism and can cover all known and unknown infectious entities other than prions. However, costs, turnaround time and human background reads in complex biofluids such as plasma stand in the way of more widespread deployment. Many protocols also require separate library preparations for DNA and RNA analytes, increasing the costs for detection. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow that addresses these issues with a human background depletion kit (HostEL) and a library preparation kit that processes DNA and RNA simultaneously (AmpRE). In our analytical validation of the HostEL workflow, we were able to enrich and detect the signal from bacteria and fungi standards spiked in at physiological levels in plasma with low-depth sequencing (< 1 million reads). Our clinical validation also shows that 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19-hour iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7-hour MiniSeq platform. Significantly, our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing. In conclusion, we demonstrate that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow and can be chosen based on required turnaround times.