Advanced method for preparing cell free DNA sequencing libraries

The goal of the proposed research is to develop an improved method RealSeq DC to prepare libraries of cell free DNA cfDNA for next generation sequencing NGS cfDNAs which are found in blood and in other bodily fluids represent promising minimally invasive liquid biopsy samples for human cancer and prenatal diagnosis of fetal genetic diseases cfDNAs comprise highly fragmented double stranded DNA fragments to bp in length having single strand nicks as well as andapos and andapos end overhangs They are normally present at low concentration in biofluids In cancer patients concentration of cfDNA and level of fragmentation are positively correlated with tumor weight with the majority of cfDNA fragments being shorter than bp Analysis of tumor specific characteristics of cfDNA such as the amount of DNA its level of fragmentation and the presence of mutations and methylated residues can be utilized for cancer diagnosis and prognosis and for evaluating tumor progression and response to treatment Next generation sequencing NGS has great potential to assess these parameters However due to the high level of DNA fragmentation in cancer derived cfDNAs they cannot be efficiently incorporated into DNA Seq libraries and therefore are under detected by conventional double stranded methods of sequencing library preparation To overcome this problem we propose an advanced method that uses short ssDNA fragments prepared by denaturation of cfDNA for ligation with a single combo adapter followed by circularization of the ligation product and direct PCR amplification rather than rolling circle amplification RCA of the circular templates This method produces monomer amplicons each containing a single cfDNA sequence insert flanked by standard Illumina andapos and andapos adapter sequences The method minimizes the formation of amplicons comprising empty adapter dimers In Phase I we plan to develop enzymatic steps specific for ssDNAs demonstrate the feasibility of the RealSeq DC approach proof of concept and its superiority in sequencing DNA fragments of nt the size range which is typical for cancer specific cfDNA over the two currently available methods of DNA Seq library preparation cfDNA isolated from plasma samples from breast cancer patients will be assayed for this comparison In Phase II we will further streamline the RealSeq DC protocol for commercialization extend the protocol to identify methylated nucleotides test its reproducibility and minimize the required cfDNA input We also will increase the number of samples studied as well as the range of physiological states and diseases with which these samples are associated to evaluate the full potential of this approach and identify any limitations Cell free DNAs cfDNAs which are found in blood and in most other bodily fluids represent promising minimally invasive liquid biopsy samples for human cancer and prenatal diagnosis of fetal genetic diseases Although next generation sequencing has great potential for analysis of cfDNA for cancer diagnosis prognosis and treatment optimization limitations of conventional methods under detect the short cancer specific cfDNA fragments The novel improved method of preparing samples for sequencing proposed here is likely to improve the prospects of early noninvasive diagnosis of cancer and fetal abnormalities including identification of specific genetic defects that may be addressable by targeted therapies