Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms

• Published in: Bioengineering (Basel) Vol. 10; no. 5; p. 520
• Main Authors: Koh, Winston Lian Chye, Poh, Si En, Lee, Chun Kiat, Chan, Tim Hon Man, Yan, Gabriel, Kong, Kiat Whye, Lau, Lalita, Lee, Wai Yip Thomas, Cheng, Clark, Hoon, Shawn, Seow, Yiqi
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 25.04.2023; MDPI
• Subjects: Bioengineering; Data analysis; Dengue fever; Deoxyribonucleic acid; Diagnostic systems; DNA; DNA sequencing; DNA/RNA library preparation; Gene sequencing; Genomes; host depletion; infectious disease; Infectious diseases; liquid biopsy; metagenomic sequencing; Metagenomics; Methods; Next-generation sequencing; Nucleotide sequencing; Pathogens; Plasma; Platforms; Ribonucleic acid; RNA; Severe acute respiratory syndrome coronavirus 2; Tropical diseases; Workflow; Singapore; United States > US; metagenomic sequencing; DNA/RNA library preparation; infectious disease; liquid biopsy; host depletion
• DOI: 10.3390/bioengineering10050520
• ISSN: 2306-5354

Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 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 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.