Solid tumor biopsies are expensive and invasive, making them less than ideal for patients who are older or very young. Furthermore, with certain forms of cancer such as non-small cell lung cancer, solid tumor biopsies are not obtainable for a significant number of patients or do not have sufficient DNA to perform NGS profiling. CCGD has extensive experience with cfDNA sequencing and is familiar with the technical characteristics that make it a challenging input. We have developed and refined our library construction and hybrid capture enrichment assays to achieve greater sensitivity. We are in the process of further refining our bioinformatics analysis workflow to take advantage of unique molecular identifiers for error detection at low tumor content.
Read more about our work in cell-free DNA.
We have recently begun a project to evaluate RNA-Seq methods for FFPE-derived RNA samples. An RNA-Seq library construction method has been validated internally. Further, refinement and technical additions to the assay are ongoing, particularly with regard to developing methods for low input and poor-quality samples. Currently, we have restricted input to samples depleted of rRNA. However, future R&D efforts will be directed to using total RNA as input. Our bioinformatics team has developed a preliminary data analysis workflow that includes expression analysis and fusion transcript detection.
Long-read sequencing would be particularly well suited to detect structural rearrangements and would not require a priori knowledge of where the rearrangement may have occurred. We have conducted a preliminary evaluation of the Oxford MinION and were able to achieve read lengths as long as 50,000 bps. Library prep and sequencing is straightforward. However, bioinformatic analysis tools are limited, although there have been significant strides within the Nanopore community. Sequencing quality is problematic for homopolymer regions and artifactual indels are a systemic issue, both of which tend to restrict usage for SNV calling. The platform is not robust enough for clinical use and can be finnicky for research purposes, but it holds a great deal of promise.
One issue that is still being resolved is how to enrich for specific regions. Conventional hybrid capture baits are limited to pulling down fragments whose length is ~2 Kb or less. Winston Timp at Johns Hopkins and Oxford Nanopore are both attempting to use Cas9 to bind and pull down large fragments of ~10 Kb. Results are promising but the method is still under development.
CCGD: Aaron ThornerProfile: Elizabeth GarciaBioinformatics: Matthew Ducar
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