PCR continues to revolutionize molecular diagnostics. Its ability to enrich genomic regions of interest for next-generation sequencing (NGS) and interface with other technologies such as mass spectrometry are helping move the field closer to the goal of personalized medicine.
CHI’s recent “Next Generation Diagnostics Summit” highlighted a number of advances in R&D in this arena.
FFPE samples represent one of the most abundant sources of readily available specimens from which to mine genetic information. But, they also are challenging to analyze via NGS. Elizabeth Mambo, Ph.D., senior scientist, technology development, Asuragen, reported on the company’s work to address the needs for mutation detection in such technically challenging samples.
“NGS platforms are an indispensable tool for deep sequencing of patient tissue samples. But FFPE and fine needle aspiration (FNA) specimens are particularly difficult to work with due to their chemical alteration (FFPE) or limited amounts (FNA and FFPE). We developed multiple PCR-based target-enrichment methods to assess such samples and have generated data from over 170 FFPE and FNA specimens utilizing different enrichment methods and NGS platforms.”
For screening purposes, the company says it utilized the RainDance RDT 1000 (RainDance Technologies), with its massively parallel picoliter droplet PCR for enrichment of up to 20,000 genomic regions, and Illumina’s Genome Analyzer that allows large-scale gene profiling or genome-wide discovery at maximal sensitivity.
For more focused screening, or confirmatory purposes, they employed the Ion Torrent PGM (Life Technologies) to achieve 3–5 million reads in a 2 hour run time. The company has also recently acquired a MiSeq (Illumina) for similar studies.
“To date, we’ve developed three PCR-based enrichment panels,” said Gary Latham, Ph.D., vp of research and technology development. “SuraSeq™ 7500, which represents more than 7,500 distinct mutations across 52 cancer genes and interrogates over 120,000 unique bases from DNA inputs as low as 250 nanograms; and SuraSeq 200 and 500, which are ideal for high-throughput, focused sequencing in commonly mutated cancer genes and require only 10–40 nanograms of DNA.”
“It is important to use a stable of instruments that rely on different sequencing chemistries,” Dr. Latham advised. “Depending on the panel size, there can be many hits, so validating positives on an orthogonal platform provides much greater confidence in the results.
“This issue is particularly critical for high-depth (>1,000x) amplicon sequencing. There are no suitable algorithms in the public domain to properly analyze such data, so we created a customized pipeline to achieve a sensitivity of 4–5% variant.”
The company will continue developing off-the-shelf and custom panels, as well as pursue implementation in its CLIA-certified laboratory.