Uniform length DNA for paired end nextgen sequencing via in vitro packaging
Small Business Information
SCARAB GENOMICS, LLC
SCARAB GENOMICS, LLC, 1202 Ann St, MADISON, WI, 53713
AbstractDESCRIPTION (provided by applicant): We have recognized an urgent need for a commercially applicable high throughput way to isolate pure DNA with long and uniform fragment length for nextgen genomic sequencing from precious samples. The advent of nextgen sequencing has driven the cost of DNA sequencing down so that soon it will be feasible to sequence the genomes of individual patients for clinical use. Thus the dream of personalized genomic medicine may soon be realized. Whole genome sequencing of patients' DNA could become a routine part of clinical practice. Germline sequences obtained early in the life of a person can be used for individualized preventive medicine, whereas, biopsy sequences taken over the course of a cancer progression will provide a guide for personalized treatment regimens. Additionally, somatic sequences of immune tissue will provide insight into the immune status of patients, including a record of previous exposure to infectious agents. The goal of this proposal is to investigate an innovative new method of preparing DNA libraries from any source that could be used by any of the current nextgen sequencing technologies. The requirements for this method are: (1) The process must efficiently convert input DNA into uniform length fragments with random endpoints, (2) The DNA should be compatible with paired end sequencing and long enough to span repeats in the human genome. This means that the DNA should be readily ligatable into circles and larger than 10 kbp. (3) The process must be readily integrated into the high throughput pipelines so that it is commercially feasible. Our method is based on one of the few biological processes that measure the length of DNA, in vitro headful packaging by the generalized transducing bacteriophage, P22. P22 can package DNA indiscriminately, producing long fragments with an extremely tight size distribution, 42,618 780 bp. Moreover, the ends are enzymatically generated meaning they are exquisitely ligatable, ideal for nextgen paired end sequencing. Once packaged, the phage particle provides a containerized cargo system for isolating the size fractionated DNA. The process entails 1) packaging a fixed length of the target DNA by the headful mechanism, (2) purifying the filled phage particles away from contaminants, (3) releasing the homogeneous length DNA from the capsids, (4) cyclizing the DNA and capturing the novel joint, (5) nextgen sequencing these paired end reads, and (6) assembling the sequence taking advantage of the long constant distance between paired end reads to span repeat regions and aid in correct assembly. To demonstrate the feasibility of our method we have successfully in vitro packaged and recovered exogenously added yeast DNA and in an independent experiment we have sequenced the 43 kb packaged DNA from two P22 lysates using Illumina paired end sequencing technology. Research proposed here will be aimed at: 1) produce an E. coli based lysogen system for production of in vitro packaging cell extracts, 2) optimize conditions for the production of 43 kb circles from packaged DNA. PUBLIC HEALTH RELEVANCE: The age of affordable personal genomics is upon us, driven by dramatic advances in nextgen sequencing technologies, and even at this early stage, the potential exists to diagnose an illness based solely on the affected tissue's genome sequence. In this proposal, we describe a powerful new method that increases the effectiveness with which genomic DNA can be processed, allowing samples as small as a biopsy to be fully sequenced and analyzed. The potential impact this technology will have on diagnosis, staging and treatment of human disease is immense.
* information listed above is at the time of submission.