NEW YORK – After launching a sequencing test for SARS-CoV-2 with Emergency Use Authorization this summer, Illumina has branched further into respiratory pathogen detection with two recently-launched research-use assays — a virus-only respiratory panel test as well as a broad diagnostic test for respiratory pathogens created in partnership with IDbyDNA.
At the Association for Molecular Pathology annual meeting, held virtually this week, Illumina provided details on its SARS-CoV-2 test, called COVIDSeq, as well as on a panel called Respiratory Virus Oligo Panel 2, or RVOP 2, that it launched earlier this year and on a platform called Explify Respiratory Pathogen Infectious Disease and Antimicrobial Resistance Panel, dubbed RPIP, developed with its partner IDbyDNA.
In an AMP workshop sponsored by Illumina, Kelly Hoon, an executive sales specialist at Illumina specializing in microbiology, described the three respiratory offerings.
For SARS-CoV-2, Illumina obtained Emergency Use Authorization for COVIDSeq in June. It was the first sequencing-based test to get EUA, with the EUA amended in August to include additional instruments and workflow tweaks.
The EUA workflow involves ribosomal RNA depletion with an Illumina product called Robo-Zero Plus, followed by Illumina Stranded Total RNA Prep Ligation, and sequencing on the NextSeq 500 and 550, as well as the 550Dx in research use only mode, Hoon said. The August EUA amendment also added the option of utilizing the high-throughput NovaSeq instrument as well as additional flow cell size options.
The test can be run using automated RNA extraction from Zymo Research, and in order to meet demand, Illumina has secured a contract with Zymo to ensure availability of the extraction kits, Hoon said. She also noted that Illumina has five partners for library preparation automation, namely Beckman Coulter, Hamilton, Eppendorf, PerkinElmer, and Tecan.
Under the EUA, the COVIDSeq test uses the Dragen analysis pipeline to address up to 3,072 samples per hour. The test deploys 98 viral amplicons tiled across the virus and 11 human mRNA internal controls. Its maximum throughput is 6,144 samples per day on the NovaSeq 6000 instrument.
Illumina had previously described a Respiratory Virus Oligos Panel, which contained hybridization capture probes to detect SARS-CoV-2 and 35 other viral pathogens, including flu strains and other human coronaviruses.
Hoon said the next generation of the test, RVOP 2, detects more than 40 viruses — including SARS-CoV-2 — using 7,800 probes, with human RNA probes for an internal control. The viral genome tiling also enables full genome recovery for subsequent strain typing and tracking viral evolution, she said.
In addition, the test utilizes a new type of library prep, called Illumina RNA Prep for Enrichment that uses bead-linked transposome, or BLT, technology to reduce enrichment time from multiple days down to nine hours, Hoon said.
"These libraries can then be run on any one of the benchtop sequencers, with multiplexing from 25 to 384 samples per run, with a run time between 17 and 21 hours," she said.
The RVOP 2 can be analyzed with the Dragen RNA Pathogen Detection analytics or with a software product from IDbyDNA called the Explify Respiratory Viral Oligo Panel Platform.
Hoon noted that superinfections and co-infections were documented in 22 percent of COVID-19 cases in a JAMA study, and that the targets in that study included many of the same viruses detected in Illumina's RVOP 2. Illumina has found the RVOP 2 detects as few as two copies of SARS-CoV-2, with enrichment accounting for improved sensitivity.
Hoon also highlighted the Explify Respiratory Pathogen ID/AMR Panel, or RPIP, which consists of an enrichment kit and software from IDbyDNA and runs using Illumina library prep and sequencing platforms.
Using a single library preparation step for RNA and DNA, targeted sequencing, and proprietary software, the test can detect and differentiate 180 bacteria, 42 viruses, 53 fungi, and 1,218 antimicrobial resistance genes, with 10 spike-in control options. Overall, the test detects 280 pathogens and can give information about resistance to more than 16 classes of antibiotics.
When run on benchtop sequencers, the panel has a turnaround time of about 24 hours, with a per-test cost comparable to PCR, according to Illumina and IDbyDNA.
Robert Schlaberg, cofounder and chief medical officer at IDbyDNA, also debuted the Explify RPIP test at the IDWeek meeting last month. In an interview, Schlaberg said that, to his knowledge, this is the first commercial sequencing test to use targeted enrichment to detect a broad range of respiratory pathogens.
The targeted enrichment approach is faster and cheaper than a shotgun metagenomics approach, he said. "Because you enrich for the informative targets, you are sequencing less of what is not informative, and so you don't need to sequence as deep or generate as many sequencing reads."
The recommendation for the Explify RPIP is to generate about 1 million reads per sample, as opposed to a shotgun test that requires somewhere between 10 million and 50 million reads, Schlaberg said.
Many of the respiratory viruses, including SARS-CoV-2, have RNA-based genomes. Schlaberg said the library prep step in the Explify kit extracts and prepares both RNA and DNA and generates a library from the cDNA and DNA.
"This is one of the innovative parts of this product, because traditionally, this would have required generation of two separate libraries," he said. "Here, this is all merged into one workflow, which reduces cost and the number of steps."
That said, the libraries generated in this workflow are also "essentially shotgun libraries," Schlaberg said, and so could be sequenced by the end users as is using a metagenomics approach, in addition to enrichment or as a reflex test, for example.
The bead-based capture step used to require overnight processing. Now, "this can be done in 24 hours, you can get both RNA and DNA targets in one approach, [and] the costs have come down ... comparable to PCR panels," Schlaberg said.
He said the kit comes as a bundle including everything needed to prepare samples for sequencing and the analysis software. "It does not include the actual sequencing reagents, and that is because you can use different Illumina sequencers for it," he said, noting that customers could use the MiniSeq for faster turnaround, or the NextSeq for higher throughput.
The test can be used to identify pathogens, but Schlaberg said that assays can also be designed to quantify pathogens, genetically profile them, or to gain further insights into antimicrobial susceptibility. The Explify RPIP was also designed to yield whole-genome sequences for SARS-CoV-2 and influenza. "This technology is very powerful beyond just broad organism identification," he said.
Importantly, infectious disease testing usually demands rapid turnaround times, and desktop sequencers, like the MiniSeq, can run in about five hours instead of the day or more typically required. "It makes a substantial difference if you are in the microbiology space," Schlaberg said.
The cost of Explify RPIP is in the same range as commercially available multiplex PCR panels, he said, or somewhere in the $100 to $200 range.
At IDWeek, Schlaberg also said that IDbyDNA is developing an Explify assay for urinary tract infections using targeted metagenomics to detect more than 150 UTI-causing pathogens and 750 AMR markers.
A sequencing approach has advantages over multiplex PCR panels in certain infectious disease cases, he said. For example, transplant patients are often given immunosuppression that leaves them vulnerable to rare respiratory pathogens which may not be represented on PCR panels.
Schlaberg said Explify is particularly useful in these patients, who can develop severe disease and can also be more susceptible to co-infections in the hospital. With targeted sequencing, "you do a broad sweep, you get all of the answers with one approach," he said. Other patients who may benefit are seriously ill people in intensive care and very young children, he added.
On the other hand, PCR panels reportedly have an advantage over sequencing in terms of the skill and time it takes to run them.
Alex Greninger, a virologist at the University of Washington, authored a review of sequencing-based approaches to infectious diseases in 2018 that noted cost is a major barrier.
In an email, Greninger said that even with reduced cost, the labor requirements of sequencing are greater than for PCR panels. "Multiplex PCR hands-on time is like 1 to 2 minutes. It's bonkers," he said. Greninger described the Explify approach as "interesting," but added "I just think it will be a while before that is a preferred method of detection."
Getting regulatory approval for sequencing-based infectious disease diagnostic tests has also been historically challenging. Schlaberg said the team is carefully considering regulatory submission for Explify RPIP, including potentially seeking approval for a narrow version of the test.