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PLoS One. 2019 Jan 10;14(1):e0206194. doi: 10.1371/journal.pone.0206194. eCollection 2019.

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

Author information

1
UCSF School of Medicine, Benioff Children's Hospital, Department of Pediatrics, Division of Critical Care, San Francisco, California, United States of America.
2
UCSF School of Medicine, Department of Biochemistry & Biophysics, San Francisco, California, United States of America.
3
Chan Zuckerberg Biohub, San Francisco, California, United States of America.

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.

PMID:
30629604
DOI:
10.1371/journal.pone.0206194
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Conflict of interest statement

The authors have declared that no competing interests exist.

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