Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing

Nguyet Kong, Whitney Ng, Kao Thao, Regina Agulto, Allison Weis, Kristi Spittle Kim, Jonas Korlach, Luke Hickey, Lenore Kelly, Stephen Lappin, Bart C Weimer

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Background: The PacBio RS II provides for single molecule, real-time DNA technology to sequence genomes and detect DNA modifications. The starting point for high-quality sequence production is high molecular weight genomic DNA. To automate the library preparation process, there must be high-throughput methods in place to assess the genomic DNA, to ensure the size and amounts of the sheared DNA fragments and final library. Findings: The library construction automation was accomplished using the Agilent NGS workstation with Bravo accessories for heating, shaking, cooling, and magnetic bead manipulations for template purification. The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated. Conclusions: Automated protocols of PacBio 10 kb library preparation produced libraries with similar technical performance to those generated manually. The TapeStation System proved to be a reliable method that could be used in a 96-well plate format to QC the DNA equivalent to the standard Bioanalyzer System results. The DNA Integrity Number that is calculated in the TapeStation System software upon analysis of genomic DNA is quite helpful to assure that the starting genomic DNA is not degraded. In this respect, the gDNA assay on the TapeStation System is preferable to the DNA 12000 assay on the Bioanalyzer System, which cannot run genomic DNA, nor can the Bioanalyzer work directly from the 96-well plates.

Original languageEnglish (US)
Article number27
JournalStandards in Genomic Sciences
Volume12
Issue number1
DOIs
StatePublished - Mar 23 2017

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Bacterial Genomes
Automation
Libraries
DNA
tetrachloroisophthalonitrile
Library Automation
Quality Control
Heating
Software
Molecular Weight

Keywords

  • Automation
  • Bacterial genomic DNA
  • Bioanalyzer
  • NGS workstation
  • PacBio SMRTbell NGS library preparation
  • TapeStation System

ASJC Scopus subject areas

  • Genetics

Cite this

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing. / Kong, Nguyet; Ng, Whitney; Thao, Kao; Agulto, Regina; Weis, Allison; Kim, Kristi Spittle; Korlach, Jonas; Hickey, Luke; Kelly, Lenore; Lappin, Stephen; Weimer, Bart C.

In: Standards in Genomic Sciences, Vol. 12, No. 1, 27, 23.03.2017.

Research output: Contribution to journalArticle

Kong, N, Ng, W, Thao, K, Agulto, R, Weis, A, Kim, KS, Korlach, J, Hickey, L, Kelly, L, Lappin, S & Weimer, BC 2017, 'Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing', Standards in Genomic Sciences, vol. 12, no. 1, 27. https://doi.org/10.1186/s40793-017-0239-1
Kong, Nguyet ; Ng, Whitney ; Thao, Kao ; Agulto, Regina ; Weis, Allison ; Kim, Kristi Spittle ; Korlach, Jonas ; Hickey, Luke ; Kelly, Lenore ; Lappin, Stephen ; Weimer, Bart C. / Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing. In: Standards in Genomic Sciences. 2017 ; Vol. 12, No. 1.
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