Miniaturization and optimization of 384-well compatible RNA sequencing library preparation

• Published in: PloS one Vol. 14; no. 1; p. e0206194
• Main Authors: Mayday, Madeline Y., Khan, Lillian M., Chow, Eric D., Zinter, Matt S., DeRisi, Joseph L.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.01.2019; Public Library of Science (PLoS)
• Subjects: Acoustics; Automation; Automation, Laboratory; Biochemistry; Biology and life sciences; Biophysics; Business metrics; Cell culture; Chemical reduction; Consortia; Cost Savings; Critical care; Deoxyribonucleic acid; DNA; DNA methylation; DNA sequencing; Feasibility Studies; Gene sequencing; Genomes; Genomics; High-Throughput Nucleotide Sequencing - economics; High-Throughput Nucleotide Sequencing - instrumentation; High-Throughput Nucleotide Sequencing - methods; Human papillomavirus; Human wastes; Laboratories; Libraries; Library collections; Medicine; Medicine and Health Sciences; Metagenomics - economics; Metagenomics - instrumentation; Metagenomics - methods; Methods; Microchemistry - economics; Microchemistry - instrumentation; Microchemistry - methods; Microorganisms; Miniaturization; Next-generation sequencing; Optimization; Pathogens; Pediatrics; Reagents; Research and analysis methods; Ribonucleic acid; RNA; Sequence Analysis, RNA - economics; Sequence Analysis, RNA - instrumentation; Sequence Analysis, RNA - methods; Tips; United States > US; San Francisco California; California
• DOI: 10.1371/journal.pone.0206194
• ISSN: 1932-6203

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.