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.
In this poster nanocrystals are formed using AFA to aid in nucleation control, and control particle size to achieve tight distribtution of 150nm particles ‘ground up’.
Continuous flow AFA-Nanocrystal™ production using a solvent/antisolvent method from small 2mL batch to a continuous flow process enabling both nano and micron sized particle production.
truCOLLECT™, a novel blood collection, stabilization, and DNA extraction system specifically designed for use in NGS
Nucleic acid-based diagnostics are rapidly moving toward next-generation sequencing (NGS). Venipuncture blood is commonly the sample of choice for high quality DNA purification, but the logistics of blood collection, stabilization, shipping/storage, and DNA extraction remain complex. Current blood collection methods require a professional phlebotomist, leak-proof overnight shipping on ice or cold-packs, and a time-critical labor-intensive DNA extraction workflow. Collectively these complexities create logistical and financial barriers for patients, researchers and clinicians. Dried blood holds the potential to overcome these barriers, however, the currently variable collection, variable stabilization, and unreliable DNA extraction, all of which affect yield and
quality, has prevented its adoption for most NGS-based applications.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.