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.
Rapid identification of infectious organisms by the clinical microbiology laboratory is critical for reducing the risk of morbidity and mortality in patients.
“Unfortunately, current methods primarily rely on the rather cumbersome and slow method of culturing specimens and using traditional phenotypic analysis for characterizing pathogens. First-generation molecular diagnostics provided greater speed, but could only assay for one or at best a few pathogens, meaning that diagnosticians often had to guess which pathogen to test for,” noted Garth D. Ehrlich, Ph.D., executive director, center for Genomic Sciences, Allegheny-Singer Research Institute.
Dr. Ehrlich says a new method coupling PCR and mass spectrometry could revolutionize the way clinical labs identify pathogens.
“We have begun using a PCR-based mass spectrometry method known as PCR-electron spray ionization, time-of-flight, mass spectrometry (PCR-ESI-TOF-MS). This technology provides unparalleled sensitivity, accuracy, and breadth within the realm of molecular diagnostics for pathogen detection. It takes advantage of the exquisite sensitivity of both PCR and mass spectrometry.
“Further, global specificity derives from the use of multiple, independent genomic amplification targets. Not only can single assays detect and speciate all members of a taxonomic domain, such as eubacteria, fungi, etc., but they can do so in hours, not days.”
The instrument used by Dr. Ehrlich and colleagues is Abbott Laboratories’ Ibis T-5000 system, predecessor to the current PLEX-ID platform that is being commercialized for clinical diagnostics. He says one doesn’t have to initially guess which pathogen(s) might be causing an infection; rather the system is capable of a broad-based identification of bacteria and fungi. His studies have evaluated samples from numerous infectious and inflammatory conditions including total joint failures, osteoarthritis, chronic nonhealing wounds, and surgical site infections, among others.
“The system can semiquantitatively identify mixed populations of microbes with a readout of genotypic analysis. There are so many advantages of this PCR-mass spec system over the rather archaic method of culturing a sample.
“Use of internal calibrations provides for relative quantitative assessments among co-infecting species. It can work even following antibiotic administration. It simultaneously detects multiple species (even biofilms) and has been shown to have superiority to all other bacterial detection methods. Also, there is no need to develop new assays for each specimen type because the technology is already established and validated.”
Although the costs per tests are not high, they do necessitate the purchase of a dedicated mass spectrometer and software package. “Despite this challenge, this new technological approach has the potential to supplant the foundation of clinical microbiology and, in so doing, save many lives.”