The most commonly used approach for fragmenting genomic material utilizes mechanical shearing. This method is advantageous in that it introduces little to no bias as a result of processing. However, mechanical shearing methods necessitate the use of specialized equipment and consumables and, currently, require several manual transfer steps for processing. The workflow presented here eliminates the need for additional transfer steps, reducing the possibility for sample loss and decreased process yield. Additionally, this workflow addresses many of the inconveniences currently associated with gDNA fragmentation on Covaris Focused-ultrasonicators.
We present a new DNA shearing vessel, the Covaris oneTUBE™ that is optimized for low DNA input and allows single tube DNA shearing and NGS library preparation. This new engineered polymer vessel incorporates features that effectively control acoustic cavitation to enable reproducible and precisely tuned hydrodynamic shear forces. Its low binding surface guarantees high DNA recovery as well as ease of volume handling down to 10 μl and picogram DNA inputs. Additionally, this vessel is fully compatible with magnetic separation procedures, thermal cyclers and liquid handling platforms. oneTUBE design is adaptable for scaled-up SBS formats, such as 96, 384, and higher well densities.
We present data that demonstrates precise and accurate DNA shearing in the Covaris oneTUBE, and validation using a wide range of DNA input quantities and %GC.
While most NGS automation workflows have focused on Library Preparation and Target Enrichment protocols, there have been only a few commercial solutions presented around the initial DNA sample transfer from the Covaris microTUBE consumables. Here we introduce an automated workflow for extracting sheared DNA samples from a 96 microTUBE Plate and 8 microTUBE Strip using the Agilent Bravo NGS liquid handling platform. We show the successful transfer of samples from each microTUBE product and demonstrate that sample and data qualities are comparable between manual and automated handling. Automation is a mandatory requirement for any high-throughput project that reduces variability, improves workflow efficiency and increases walkaway time. Utilizing the Agilent Bravo Liquid Handling Platform for automating extraction of sheared DNA from the Covaris 96 microTUBE Plate and 8 microTUBE Strip offers a reliable and reproducible solution for high-throughput studies.
Background: Cystic fibrosis (CF) is a genetic disorder that afflicts over 70,000 people worldwide. It is characterized by viscous mucus that accumulates in the lungs creating challenges for microbiology labs processing sputa toisolates lower growing fungal species. Our laboratory attempted to liquefy and homogenize mucoid sputum samples by exposure to Adaptive Focused Acoustics™ (AFA) in order to increase recovery of fungal pathogens.
Poster presented at ASHG2015 – Here, we present two innovations that together overcome many of the drawbacks of the current DBS technology: (1) a novel sampling device for collecting capillary blood (truCOLLECT™) that provides integrated sample barcoding, eliminates the need for a drying period before shipment, and yields DNA and protein amounts similar to those seen with fresh blood; and (2) use of Adaptive Focused Acoustics (AFA™) for efficient release of biomarker from truCOLLECT™ devices as well as from conventional DBS (truXTRAC™ DBS). The AFA™-based extraction process is highly automated and allows batch processing with less than 30 min hands-on-time per 96 samples.
We have evaluated the Adaptive Focused Acoustics (AFA™) system by Covaris as a method for obtaining high molecular weight DNA suitable for SMRTbell template preparation and subsequent single molecule sequencing. Using this method, genomic DNA was extracted from normal kidney FFPE scrolls acquired from Cooperative Human Tissue Network (CHTN), University of Pennsylvania. Damaged sites present in the extracted DNA were repaired using a DNA Damage Repair step, and the treated DNA was constructed into SMRTbell libraries suitable for sequencing on the PacBio RS II System. Using the same repaired DNA, we also tested PCR efficiency of target gene regions of up to 5 kb. The resulting amplicons were constructed into SMRTbell templates for full-length sequencing on the PacBio RS II System. We found the Adaptive Focused Acoustics (AFA) system combined with truXTRAC™ by Covaris to be effective and efficient. This system is easy and simple to use, and the resulting DNA is compatible with SMRTbell library preparation for targeted and whole genome SMRT Sequencing. The data presented here demonstrates single molecule sequencing of DNA samples extracted from tissues embedded in FFPE.
Primo Baybayan, et al
Poster presented at AGBT 2016 – truCOLLECT™, a novel blood collection, stabilization, and DNA extraction system specifically designed for use in NGS
In this poster, we introduce truCOLLECT™, a novel blood collection system which does not require a venous blood draw and greatly simplifies blood collection, standardizes dry stabilization, and simplifies shipment while delivering DNA yield and quality comparable to fresh whole blood.
Kakumanu, Schmitt, He, Beckett, Laugharn.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.