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.
Highlights
Metagenomic next-generation sequencing is becoming an increasingly useful tool in the field of biology and clinical medicine
Miniaturization and optimization of 384-well compatible RNA sequencing library preparation per-base cost of sequencing has become less expensive over the last several decades, the cost and time associated with sample preparation remain disproportionately high [1,2]
Though these provide more hands-off time during the library preparation process, the overall cost can often exceed that of hand-prepared libraries due to the increased dead volume of reagents and the large number of expensive, sometimes proprietary tips required for liquid handlers
Summary
Metagenomic next-generation sequencing (mNGS) is becoming an increasingly useful tool in the field of biology and clinical medicine. Numerous library preparation protocols have been adapted for automation through the use of various positive displacement tip-based liquid handler instruments, including the Beckman Coulter Biomek, Hamilton Star, Agilent Technologies Bravo, TTP LabTech Mosquito, and others [3,4,5]. Though these provide more hands-off time during the library preparation process, the overall cost can often exceed that of hand-prepared libraries due to the increased dead volume of reagents and the large number of expensive, sometimes proprietary tips required for liquid handlers. We describe a detailed protocol that provides high-fidelity, miniaturized, automated, cost- and time-efficient 384-well library preparation together with its quality and performance metrics
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