Stephen Lappin’s research while affiliated with Agilent and other places

Figure 1: 100K Pathogen Genome Project sample preparation workflow for multiplexed, short-read Illumina sequencing Figure 3: Detailed KAPA HTP Library Preparation protocol. The input into library construction is fragmented DNA or cDNA. Each enzymatic reaction is followed by a SPRI-bead cleanup. The "with-bead" protocol uses a single aliquot of SPRI beads for all cleanups prior to library amplification, producing higher yields of adapter-ligated libraries, and reduces the number of amplification cycles to generate sufficient material for Library QC and sequencing. Figure 2: Representative electropherograms of Listeria (generated on the Agilent 2100 Bioanalyzer system and Agilent 2200 TapeStation system) of bacterial libraries prepared for whole genome sequencing with the KAPA HTP Library Preparation Kit. The average library size for each genus was as indicated. Peaks at 35 and 10381 bp are internal standards used for alignment and quantitation determination with the Agilent 2100 Bioanalyzer system. ABSTRACT A method was developed to automate the KAPA HTP Library Preparation kit for microbial whole genome sequencing. This method uses the Agilent NGS Workstation, consisting of the NGS Bravo liquid handling platform with its accessories for heating, cooling, shaking, and magnetic bead manipulations in a 96-well format. User intervention in multistep protocols is minimized through the use of other components of the workstation such as the BenchCel 4R Microplate Handler and Labware MiniHub for labware storage and movement. This method has been validated for sequencing on the Illumina platform and consists of three protocols: the first is for end repair to post-ligation cleanup; the second is used for library amplification setup; and the third is for the post-amplification cleanup. The modular design provides the end-user with the flexibility to complete library construction over two days, and is suitable for the construction of high-quality libraries from bacteria of various GC content. This combined solution produced a workflow that is suitable for production-scale sequencing projects such as the 100K Pathogen Genome Project.

The PacBio RS II provides for single molecular, real-time (SMRT) DNA technology to sequence genomes and detect DNA modifications. The quality control methods from gDNA input to the final library using the Agilent BioanalyzerSytem and Agilent TapeStation System were evaluated. 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 TapeStationSystem 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 BioanalyzerSystem, which cannot run genomic DNA, nor can the Bioanalyzer work directly from the 96-well plates.

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. 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. 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.

Automation of the PacBio 10 kb Template Preparation using the SMRTbell Template Prep kit for the sequencing of bacterial genomes on the Agilent Automated Liquid Handling Platform is described. Four bacterial genomes of differing GC content are used to prepare libraries without bias reading for next generation sequencing. The gDNA quality assessment was done using spectrophotometric and automated elec-trophoresis for high molecular weight DNA with an Agilent 2200 TapeStation system with the Agilent Genomic DNA ScreenTape assay. The sheared DNA was measured with the Agilent Bioanalyzer system and the DNA 12000 Kit.

A new method was developed to automate the KAPA HTP Library Preparation kit for microbial whole genome sequencing. This method uses the Agilent NGS Workstation, consisting of the NGS Bravo liquid handling platform with its accessories for heating, cooling, shaking, and magnetic bead manipulations in a 96-well format. User intervention in multistep protocols is minimized through the use of other components of the workstation such as the BenchCel 4R Microplate Handler and Labware MiniHub for labware storage and movement. This method has been validated for sequencing on the Illumina platform and consists of three protocols: the first is for end repair to post-ligation cleanup; the second is used for library amplification setup; and the third is for the post-amplification cleanup. The modular design provides the end-user with the flexibility to complete library construction over two days, and is suitable for the construction of high-quality libraries from bacteria of various GC content. This combined solution produced a workflow that is suitable for production-scale sequencing projects such as the 100K Pathogen Genome Project.