|Education||BS, Duke University|
Extensive knowledge of the genetic alterations that underlie cancer is now available, opening new opportunities for the management of patients. Some of the most important of these opportunities involve “liquid biopsies”, i.e., the evaluation of blood and other bodily fluids for mutant DNA template molecules that are released from tumor cells into such fluids. It has recently been shown that liquid biopsies in blood can detect minimal amounts of disease in patients with early stage colorectal cancers, thereby providing evidence that could substantially affect their survival. Other studies have shown that circulating tumor DNA (ctDNA) can be detected in the blood of patients with other malignancies.
The most powerful of these approaches to detecting mutant DNA template molecules employs massively parallel sequencing to simultaneously analyze the entire sequences of hundreds of millions of individually amplified template molecules. However, all of the currently available sequencing instruments have relatively high error rates, limiting sensitivity at many positions to one mutant among 100 wild type template molecules, even with DNA templates that are of optimal quality. The DNA quality of clinical samples is often far less than optimal, compounding the problem.
An important way to improve sensitivity is with the use of “molecular barcodes”, in which each template is covalently linked to a unique identifying sequence (“UID”) prior to the final amplification round. Molecular barcodes were originally used to precisely count individual template molecules, but were subsequently discovered to provide a powerful approach, termed SafeSeqS, for error reduction. The final amplification round produces multiple copies of each UID-linked template. Each of the daughter molecules produced by amplification contains the same UID, forming a “UID family”. To be considered a bona fide mutation every member of the UID family must have the identical sequence at each queried position.
There are two general ways to assign molecular barcodes to template DNA molecules. One is used to PCR-amplify specific loci using a set of locus-specific primers and the other used to ligate adapters prior to amplification of the entire genome, creating a library. The PCR method uses primers containing a stretch of random bases to distinguish each individual template molecule. The advantage of this approach is that it is applicable to very small amounts of DNA and virtually the only sequences amplified are the desired ones, reducing the amount of sequencing needed to evaluate a specific mutation. The disadvantage is that errors introduced into one strand before the final PCR cycle will create its own UID family, mimicking a supermutant. The ligation method either employs random sequences in the adapters used for ligation, or uses the ends of the randomly sheared template DNA to which the adapters are ligated as endogenous UIDs. However, errors are still introduced during the PCR steps with this approach. My current thesis project focuses on improving the specificity and sensitivity of next generation sequencing to increase the confidence in detecting rare mutations in clinical samples. Increased confidence will allow for more robust cancer screening in patients, as well as allow for in-depth profiling of small subsets of cells within a primary tumor that will eventually give rise to metastasis and systemic disease.