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

Abstract

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.

Pan-viral serology implicates enteroviruses in acute flaccid myelitis

“…RNA sequencing libraries were prepared using a previously described protocol optimized and adapted for miniaturization and automation. 29 Libraries were sequenced on a NovaSeq 6000 machine (Illumina) to generate 150 nucleotide (nt), paired-end reads. Samples were also sequenced after enrichment for EV-A71 and EV-D68 genomes using FLASH (Finding Low Abundance Sequences by Hybridization, Supplemental Table 2).…”

Clinical features, diagnostics, and outcomes of patients presenting with acute respiratory illness: a comparison of patients with and without COVID-19

“…26 The latter enabled subsequent bioinformatic assessment of the total RNA mass input in each sample. 27 RNA was then fragmented and subjected to a modified metagenomic spiked sequencing primer enrichment (MSSPE) library preparation method. 28 Briefly, a 1:1 mixture of the NEBNext Ultra II RNAseq Library Prep (New England Biolabs) random primer stock and a pool of SARS-CoV-2 primers at 100 µM was used at the first strand synthesis step of the standard RNAseq library preparation protocol to enrich for the recovery of reads spanning the length of the SARS-CoV-2 genome sequence in the context of mNGS analysis.…”

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

“…Researchers may also decrease the cost through reducing library preparation reaction volumes, as this is typically the most costly step in NGS preparation (Table S10). While reducing reaction volumes deviates from the formulations validated by manufacturers, many researchers (including ourselves) have used half-reactions for preparing NGS libraries with no noticeable effect on quality, and other studies have reported reliable library preparation down to one-sixteenth reactions (44–47).…”