Abstract
BackgroundThe 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.FindingsThe 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.ConclusionsAutomated 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.
Highlights
Increased throughput from the use of generation sequencing methods has revealed new information about the function and structure of bacterial genomes
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
Summary
Increased throughput from the use of generation sequencing methods has revealed new information about the function and structure of bacterial genomes. The use of short reads to produce draft genomes leads to problems with GC content bias and repeat regions that make it tedious to produce closed genome assemblies. This technical note discusses the PacBio RS II approach using a single molecule, real-time DNA sequencing approach to improve genome assembly through extra-long read lengths. The 100K Pathogen Genome Project [1] is using the PacBio 10 kb SMRTbell Template Preparation kit to produce 1,000 closed genomes The scale of this project required automation of the construction of the sequencing (SMRTbellTM) library. 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
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