SBIR-STTR Award

The ability to sequence and map long stretches of DNA using third generation sequencing technologies is set to transform the field of genomics and allow researchers and clinicians unparalleled insight into the complexities of our genome and diseases. However, the move to these platforms presents new hurdles in long DNA sample preparation. Most conventional spin column sample preparation methods shear DNA during purification. And, although agarose plug digestion methods can isolate ultra-long DNA from samples, the protocols can be difficult, time consuming and highly variable. While newer technologies have emerged that allow for more rapid extraction of long-DNA these platforms are limited in starting sample type to fresh blood or tissue culture cells and exclude use of most clinically relevant frozen blood and solid tissue samples. At Claremont BioSolutions (CBIO), we are developing a novel extraction technology that utilizes a proprietary high capacity nucleic acid binding matrix, which is loosely packed in a low pressure fast flow column. Combined with CBIO's gentle upfront lysis method, the technology will be compatible with any starting tissue sample type, including frozen blood and solid tissue, and can be used to isolate ultra-long DNA in <30 minutes. In Aim 1, we will demonstrate CBIO's fast-flow isolation of high quantity, high quality, ultra-long DNA (>250 kilobases) from fresh and frozen blood and tissue samples and benchmark against existing extraction methods. In Aim 2, we will validate isolated ultra-long DNA compatibility with two third generation long-read sequencing and mapping technologies and demonstrate improved long-read length over existing DNA extraction methods. The future goal of this project will be to develop a platform that will accept a sample and automate all steps of sample lysis and ultra-long DNA extraction without user interaction. The proposed system would simplify workflow, lower cost and reduce variability.

Public Health Relevance Statement:
Narrative Claremont BioSolutions, LLC (CBIO) Fresh and archived tissue samples offer a wealth of untapped information relevant to cancer and genetic diseases. Extracting high quality ultra-long DNA from fresh and frozen blood and solid tissue samples for downstream long-read genomic applications is often difficult, labor intensive and highly variable. This project will apply a new technology to allow rapid isolation and reproducible extraction of ultra-long DNA for tissue samples for downstream NGS and long-read genomic applications.

Project Terms:
Archives; Bacteria; base; Benchmarking; Binding; biobank; Blood; Blood specimen; cancer genetics; Cells; Chromium; clinically relevant; Comb animal structure; commercialization; cost; cost effective; Cytolysis; Digestion; Disease; DNA; Engineering; Flowcharts; Fluorescent Probes; Freezing; Future; Generations; Genetic Diseases; Genome; Genomics; Goals; improved; innovation; insight; International; Length; Link; Malignant Neoplasms; Manuals; Maps; Measures; Methods; microbial; Molecular; nanopore; new technology; novel; Nucleic Acid Binding; Nucleic Acids; physical mapping; Preparation; pressure; Principal Investigator; Protocols documentation; Reproducibility; Research Personnel; RNA; Running; Sampling; Sepharose; Small Business Innovation Research Grant; Solid; space station; Stretching; System; Technology; Testing; Third Generation Sequencing; Time; Tissue Sample; tissue/cell culture; Tissues; Variant; Work; Yeasts

Claremont BioSolutions, LLC (CBIO)The ability to sequence and map long stretches of DNA and RNA using third generation sequencingtechnologies is set to transform the field of genomics and allow researchers and clinicians unparalleled insightinto the complexities of our genome and diseases. However, the move to these platforms presents newhurdles in long DNA and RNA sample preparation. Most conventional spin column sample preparationmethods shear DNA during purification, and although agarose plug digestion methods can isolate ultra-longDNA from samples, the protocols can be difficult, time consuming and highly variable. While newertechnologies have emerged that allow for more rapid extraction of long-DNA, these kits are either limited in useto easy-to-lyse sample types (cells and blood) or require extra equipment and long processing times, whichsignificantly increases the cost. At Claremont BioSolutions (CBio), we are developing a novel automatedextraction technology that will incorporate lysis, nucleic acid binding and isolation. The technology will allowusers to add and sample, hit "GO" and in 30 minutes collect DNA or RNA. In Aim 1, we will integrate our fastlysis and isolation technology into an automated cartridge to provide reproducible high quality, ultra-long DNAor RNA extraction. In Aim 2, we will validate automated ultra-long DNA extraction using easy and complexsample types, compare our technology to HMW DNA competitor's kits, and demonstrate improved long-readsequencing using two third generation technologies. In Aim 3 we will test automated RNA extraction anddownstream native RNA sequencing. In Aim 4 we will perform pre-market analysis at off-site alpha and betatesting facilities to generate end-user feedback and lockdown the design. The proposed system would simplifyworkflow, lower cost, and reduce variability.

Public Health Relevance Statement:
Narrative Claremont BioSolutions, LLC (CBIO) Isolating high quality nucleic acid from samples for downstream third generation sequencing and mapping applications is often difficult, labor intensive and highly variable. This project will automate a new technology to allow rapid isolation of ultra-long DNA and RNA from cells, tissues, and more complex samples types for downstream NGS and long-read genomic applications.

Project Terms:

<3rd generation sequencing>