Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing.
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.


RESULTS
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.
Population Health and Reproduction Department, School of Veterinary Medicine, University of California-Davis, Davis, CA USA.
Nguyet
N
Kong
Genentech, S. San Francisco, CA USA.
Whitney
W
Ng
University of California-San Francisco, San Francisco, CA USA.
Kao
K
Thao
Population Health and Reproduction Department, School of Veterinary Medicine, University of California-Davis, Davis, CA USA.
Regina
R
Agulto
Population Health and Reproduction Department, School of Veterinary Medicine, University of California-Davis, Davis, CA USA.
Allison
A
Weis
Pacific Biosciences, Menlo Park, CA USA.
Kristi Spittle
KS
Kim
Pacific Biosciences, Menlo Park, CA USA.
Jonas
J
Korlach
Pacific Biosciences, Menlo Park, CA USA.
Luke
L
Hickey
Agilent Technologies, Inc., Santa Clara, CA USA.
Lenore
L
Kelly
Agilent Technologies, Inc., Santa Clara, CA USA.
Stephen
S
Lappin
Population Health and Reproduction Department, School of Veterinary Medicine, University of California-Davis, Davis, CA USA.
Bart C
BC
Weimer
Automation; Bacterial genomic DNA; Bioanalyzer; NGS workstation; PacBio SMRTbell NGS library preparation; TapeStation System