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TapeStation to QC DNA and shearing for NGS library construction
Abstract
This application note describes the use of the Agilent TapeStation to measure DNA and sheared DNA quality for NGS of bacteria
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This Application Note demonstrates the use and application of the Agilent Genomic DNA ScreenTape assay in conjunction with the Agilent 2200 TapeStation system for easy sizing, quantification and quality assessment of genomic DNA samples. Intact, high quality genomic DNA in combination with accurate sample quantification
is required for numerous applications, particularly array comparative genome hybridization and next generation sequencing.
The initial step in Next Generation Sequencing is to construct a library from genomic DNA. To gain the optimum result, extracted DNA must be of high molecular weight with limited degradation. High-throughput sequencing projects, such as the 100K Pathogen Genome Project, require methods to rapidly assess the quantity and quality of genomic DNA extracts. In this study, assessment of the applicability of the Agilent 2200 TapeStation was done using genomic DNA from nine foodborne pathogens using several accepted high-throughput methods. The Agilent 2200 TapeStation System with Genomic DNA ScreenTape and Genomic DNA Reagents was easy to use with minimal manual intervention. An important advantage of the 2200 TapeStation over other high-throughput methods was that high molecular weight genomic DNA quality and quantity can be quantified apart from lower molecular weight size ranges, providing a distinct advantage in the library construction pipeline and over other methods available for this important step in the Next Generation Sequencing process.
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.
The Agilent 2200 TapeStation system allows for the analysis of samples in the Next Generation Sequencing (NGS) workflow from the starting material, through to final quality control (QC) of the DNA library prior to sequencing. This Application Note follows the SureSelect library preparation protocol and demonstrates the performance and applicability of the Genomic DNA ScreenTape, D1000 ScreenTape, and High Sensitivity D1000 ScreenTape assays in analyzing samples from this workflow. The data demonstrates the 2200 TapeStation system as a reliable QC platform for sizing and quantifi cation analysis of the libraries. The data also demonstrates that these DNA ScreenTape assays are comparable and within specification of the equivalent assays of the Agilent 2100 Bioanalyzer system.
Background
The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups.
Results
Up to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6%) by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes.
Conclusions
Here we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.
The Wellcome Trust Sanger Institute is one of the world's largest genome centers, and a substantial amount of our sequencing is performed with 'next-generation' massively parallel sequencing technologies: in June 2008 the quantity of purity-filtered sequence data generated by our Genome Analyzer (Illumina) platforms reached 1 terabase, and our average weekly Illumina production output is currently 64 gigabases. Here we describe a set of improvements we have made to the standard Illumina protocols to make the library preparation more reliable in a high-throughput environment, to reduce bias, tighten insert size distribution and reliably obtain high yields of data.
The Detroit Police Crime Lab has historically used Chelex as a method to isolate DNA for amplification and typing of bloodstains at the HLADQA1, PM and D1S80 loci. However, preliminary validation of several STR systems for casework has demonstrated that the Chelex procedure is not the best method of DNA isolation for STR amplifications for our purposes. Long term storage at -20 degrees C in the presence of unbuffered Chelex beads (approximately 1 year), combined with multiple freeze thaws, resulted in signal loss at a locus for many database samples. Therefore, we have employed the QIAamp spin column as an alternative method of DNA isolation for amplification and typing of STR loci currently being validated for use in the laboratory. Moreover, we determined that QIAamp isolated DNA is also suitable for HLADQA1, PM and D1S80 typing. A matrix study was performed to determine if the QIAamp DNA procedure would give better results on bloodstains deposited on "problem surfaces" such as leather, dirt and various dyed fabrics. Again, QIAamp isolated DNA was more readily typeable than Chelex isolated DNA. We successfully replaced the phenol/chloroform extraction steps utilized in our laboratory for differential extractions, a commonly used method for separating sperm and non-sperm fractions of sexual assault evidence, with the QIAamp spin columns. The QIAamp extracted DNA performed as well in all PCR amplification and typing procedures tested (PM, HLADQA1, D1S80, and STR (PowerPlex)) as the phenol/chloroform Centricon isolated or EtOH precipitated DNAs. Thus we concluded that QIAamp spin columns are a superior method for isolating DNA to be typed for a variety of loci.
Agilent Technologies
Genome Sequencing on the Illumina Platform " Agilent
Technologies, publication number 5991-4296EN, 2014.