Powerful next-generation sequencing (NGS) platforms have led to the development of large-scale genome initiatives like the 100,000 Genomes Project, The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and St. Jude Children’s Research Hospital–Washington University Pediatric Cancer Genome Project (PCGP). Sequencing at this level requires complete automation of the entire genomic workflow. Both the Fragment Analyzer semiautomated capillary electrophoresis system and the fully automated Fragment Analyzer INFINITY from Advanced Analytical Technologies, Inc. (AATI) (Ankeny, Iowa) automate the quality and quantity analysis of NGS library preparations.
Next-generation sequencing platforms
NGS platforms include systems based on sequencing by synthesis, as well as on ion and nanopore sequencing. Although these technologies have some variations in the mechanism by which sequencing is performed, they all share a common genomic DNA (gDNA) library preparation procedure. This workflow includes gDNA quality and quantity assessment, DNA fragmentation and sizing (involving mechanical shearing, sonication, nebulization or enzyme digestion), DNA repair and end polishing, platform-specific adaptor ligation and library enrichment (Figure 1). Regardless of the platform, successful sequencing requires high-quality input DNA.
Figure 1 – Stages of NGS library construction.Quantification and size determination of both unsheared gDNA and the downstream fragmented DNA is a critical aspect of the genomic workflow, but is labor intensive and historically not well-suited for complete automation. Slab gels, coupled with methods such as fluorometry or UV spectroscopy for quantification, have been used for nucleic acid sizing and quantification. Preferred methods for DNA quality and quantity measurements are capillary or microchannel electrophoresis systems. Microchannel chip-based electrophoresis units can quantify and qualify sheared DNA, but are limited in that they 1) cannot accurately size or quantify intact gDNA; 2) are difficult to automate, as new chips must be regularly loaded onto the system; 3) require manual pipetting of gels, ladders and markers onto the chips; and 4) suffer from bubbles or air gaps in the microchannels, which can lead to poor-quality separations.
Moving toward fully automated nucleic acid assessment
The Fragment Analyzer automated capillary electrophoresis (CE) system bridges the gap between manual instruments and fully automated, robotic, high-performance nucleic acid analysis. Users can place up to three 96-well plates on the system, queue up sample information and walk away. Multiple users can load and queue samples while other samples are running. The system can separate 12, 48 or 96 samples simultaneously and holds up to two different gels, so that different types of analyses may be performed for each run. For example, users can run NGS samples in one run, followed by large fragments or gDNA samples in the next run without manually changing the gel. This semiautomated instrument can analyze from 12 to 3000 samples per day, meeting the needs of most laboratories. Software allows for automated analysis and interfacing to a LIMS. High-throughput labs can configure the system so that data can be analyzed automatically at the end of a run, and the LIMS automatically ports the bar-coded plate data to appropriate locations, if desired.
The Fragment Analyzer eliminates the need for costly external robotic systems and provides various options for throughput and automation. For laboratories that demand very high throughput, the Fragment Analyzer INFINITY enables interfacing with an external robotic system.
An instrument’s degree of total automation is a measure of its walkaway capabilities, defined as how long the system can be run and serviced by a robot without human intervention. Without an external robotic interface, up to three sample plates can be loaded and run using the Fragment Analyzer, with no human intervention required. The walkaway time with three plates ranges from 1.5 to 3 hours, depending on the application.
The Fragment Analyzer INFINITY permits laboratories to operate continuously without human intervention, and thus allows for walkaway times of greater than 24 hours.
Enhanced quality and quantity assessment of gDNA fragment size
NGS employs the technique of shotgun fragmentation, in which the gDNA is sheared into smaller fragments for sequencing. For NGS, DNA shearing is controlled to obtain fragments ranging from 50 to 50,000 base pairs (bp).
Using the Fragment Analyzer, sheared DNA ranging in size from 50 to 50,000 bp can be sized using either AATI NGS Fragment Analysis Kits (1–6000 bp), the Large Fragment Analysis Kit (50–20,000 bp) or the gDNA analysis kit (1000–50,000 bp). The Fragment Analyzer also has a kit specifically designed for assessing the quality of unsheared gDNA. With capillaries up to 80 cm long, the Fragment Analyzer is also the only commercially available capillary electrophoresis instrument capable of ultrahigh resolution of small fragments, with resolution down to 3 bp for fragments under 300 bp. The Fragment Analyzer INFINITY provides ultrahigh-throughput laboratories seamless integration with an external robotic system, allowing for around-the-clock evaluation of DNA.
The quality of gDNA is assessed using a GQN, or genomic quality number. This user-defined threshold allows users to set a base pair value they consider to be good-quality DNA for a particular application. The software then calculates the percentage of gDNA above the threshold value, producing a factor of 0 to 10. Typical results for gDNA with various degrees of shearing are shown in Figure 2. A low GQN such as 2.5 represents sheared or degraded gDNA, whereas a high GQN like 9.5 represents undegraded gDNA. In Figure 2, a user-defined threshold of 45,000 bp was used.
Figure 2 – Analysis of three gDNA samples with unsheared (GQN 9.5), partially sheared (GQN 8.0) and fully sheared (GQN 2.5) DNA. The GQN is a user-defined threshold that allows researchers to define the boundary of degradation (shown with a threshold set at 48,500 bp).Conclusion
As new ultrahigh-throughput applications emerge in next-generation sequencing, laboratories will continue to push the limits of their efficiency and throughput capacity. Complete automation of DNA library assessment and automated analysis allows researchers to meet these needs.
Pierre Varineau, Ph.D., is chief technology officer, and Steve Siembieda is vice president, Commercialization, Advanced Analytical Technologies, Inc., 2450 S.E. Oak Tree Ct., Ankeny, Iowa 50021, U.S.A.; tel.: 515-964-8500; e-mail: [email protected]; www.aati-us.com