Method or test? Providing clarity to clinicians on NGS

Anne Ford

September 2016—Whether it was “This is your brain on drugs,” “Take a bite out of crime,” or “Friends don’t let friends drive drunk” popping up onscreen, few of us watching TV in the 1970s and ’80s enjoyed having our programs interrupted by those public service announcements. Yet those important messages stuck in viewers’ brains—and stuck hard, if homages such as the Washington Post’s “10 Best PSAs of All Time” are anything to go by.

John D. Pfeifer, MD, PhD, had similar hopes for his thoughts on next-generation sequencing, which he presented during a webinar produced by CAP TODAY in collaboration with Horizon’s Diagnostics Division, “Assessing the Clinical Genome: Thoughts and Lessons for Improving Diagnostic Tests” (http://bit.ly/062116_capwebinar).

The two other webinar speakers focused on the wet-bench parts of NGS and on bioinformatics, and Dr. Pfeifer said his own talk was positioned between the two. “I’m going to raise some issues that may not be as reassuring, and that may actually create some unease. . . . And so my talk today is going to be, if you will, a public service announcement.

“It’s like one of those commercials that interrupts your favorite TV show,” he said, “and it raises your awareness about something that maybe is not entirely comfortable . . . But hopefully you’re a better person at the end of it.” Dr. Pfeifer is vice chair for clinical affairs in pathology and immunology and a professor of pathology and immunology and of obstetrics and gynecology at Washington University School of Medicine.

What was this potentially uncomfortable topic? The variability of next-generation sequencing among expert laboratories, largely owing to the use of different platform and test designs, and the need to communicate more often and more openly with the clinicians who order the tests.

The pathologists and laboratory professionals who perform next-generation sequencing, he said, tend to focus on the analytic components of the sequencing, but often fail to recognize a question that creates a lot of ambiguity in the broader context of patient care: Is NGS a test or a method?

“The reason I ask that question,” he told listeners, “is to help us all remember that in the laboratory, we have a specific view about what we’re doing, but those of us who are laboratory professionals sometimes lose sight of what our clinical colleagues who are ordering the testing may or may not know.”

Pathologists and laboratory professionals tend to think of NGS as a methodology, Dr. Pfeifer said, whereas their colleagues in clinical practice often view it as a test. “In their mind, it often centers on the intended use: What is the information they hope to get out of the test?” he said. “We in the laboratory tend to think of NGS as a method where we go through all these steps . . . and we end up with a set of sequence variants through an assay that is very highly validated. . . .”

Analytically, he said, pathologists and laboratory professionals know there are “different platforms, different assay designs, different targeted genes within a panel, different mutation classes, the single nucleotide polymorphisms or single nucleotide variants, and indels, copy variants, and structural variants such as translocations. And that there are different bioinformatics pipelines that need to be optimized to find each one of those.”

In contrast, “our clinical colleagues may not recognize that NGS is not one thing,” he said.

This ambiguity and lack of communication about it can create problems, in Dr. Pfeifer’s view.

First, different NGS instruments use different chemical methods to produce the raw sequence. Some of the best examples, he said, are the Illumina HiSeq, NextSeq, and MiSeq series of instruments, which perform DNA sequence analysis via synthesis and which employ chemistry that utilizes fluorescence from incorporated bases during strand elongation.

The Thermo Fisher Ion Torrent, Ion Proton, Ion PGM, and Ion Chef series of instruments use semiconductor technology and utilize pH changes to measure incorporation of bases during strand elongation—also sequencing by synthesis.

Qiagen recently released its GeneReader NGS platform, and other platforms, such as from Oxford Nanopore, are in development.