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Although DNA sequencing has been around for several decades - many viruses (including Influenza, Ebola, and Nipah) are built from RNA - and in order to sequence them today (via RT-PCR) they must be converted from a single-stranded RNA into double-stranded complementary DNA (cDNA).
This indirect method is not only time consuming, it has the potential to introduce small errors or distortions, making the direct sequencing of RNA a highly desirable, albeit elusive goal.Today, however, we've a report in Nature Scientific Reports and a statement from the CDC on the first successful direct sequencing of viral RNA, which they hope will lead to a better understanding of flu (and other RNA) viruses.
While terrific news, some of the required technology will need to catch up and be improved in order to make this a truly useful tool. That said, this is a highly encouraging step in that direction.First today's CDC announcement, followed by a link to the full report in Nature.
(Continue . . . )
CDC Scientists Become First in History to Directly Sequence the Entire RNA Genomes of Influenza A Viruses
September 26, 2018
In a historic first, a group of CDC laboratory and bioinformatics scientists became the first to directly sequence an RNA genome. They did so with the RNA genomes of five influenza (Flu) A viruses, including seasonal influenza A and avian influenza A viruses. This work is described in an article published today in the journal Scientific Reports-Nature. This scientific achievement may shed light upon how influenza viruses function, their lifecycle, and how they change during the course of infection. Furthermore, the methods used in this study could be used to learn more about other RNA viruses, such as Ebola, HIV, measles and rabies viruses.
Information determining the makeup of nearly every living thing is stored in dual-stranded DNA. The complete set of these DNA instructions for an organism is called its “genome.” A genome is like a blueprint for how any given organism, including a person, is made. However, while the genomes of people and other living things consist of DNA, some things that aren’t technically “living,” such as viruses, have genomes coded by RNA instructions instead. Influenza viruses are an example of an RNA virus.
For decades, scientists who wanted to research the genome of RNA viruses, such as influenza, had to do so using an indirect and time-consuming method that involved first converting the single-stranded RNA into double-stranded DNA. This method, often referred to as “reverse transcription polymerase chain reaction” (RT-PCR), works well for clinical purposes, such as identifying specific viruses from respiratory samples taken from sick patients. However, scientists believe that certain small features of the virus may get lost during the conversion from RNA to DNA.
The new method described in this study has the potential to allow researchers to decode the genome of an RNA virus with greater detail (and less distortion) than ever before. For example, compare an original photograph to a copy of the same photograph. The copy will give you a pretty good idea of the original (the same can be said of RT-PCR), but the copy may lack the resolution and granularity of all the details found in the original photo.
So how does this new method work? As Matthew Keller, the first author of the paper explains, it first requires a specific machine called a “nanopore sequencer.” This machine threads a DNA or RNA strand through a tiny hole. Keller compared it to pulling a string of beads through a clenched fist. The machine then runs an electrical current across the fist, and it measures the current (as picoamps) as each bead passes through. As the machine takes these measurements, it decodes the genetic sequence of the DNA or RNA strand.
Whereas a computer decodes a series of binary numbers (i.e., ones and zeroes), DNA and RNA are encoded into a series of four letters. For DNA, these letters are A, C, G and T. For RNA, the T becomes a U, so the letters are A, C, G and U. The T stands for “Thymine,” whereas the U stands for “Uracil.” According to Keller, this is one of the main differences between DNA and RNA, and why translating RNA into DNA can sometimes result in information loss.
One capability of the nanopore sequencer is to sequence messenger RNA. Messenger RNA is a kind of intermediary that tells the body how to convert the instructions contained in the genome into actual proteins. It was this messenger RNA workflow that was modified to sequence influenza viral RNA. Keller said that messenger RNA has a tail end that is comprised of a sequences of “A’s.” By modifying the adapter that targets this region, Keller et al. were able to get the machine to specifically target and sequence flu virus RNA.
However, analyzing the data was another matter. To accomplish this, Ben Rambo-Martin with the CDC Influenza Division’s flu Informatics team also modified existing tools, but this time, they were computational tools rather than molecular ones. Rambo-Martin’s work translated the data into something that made sense, and he was able to confirm that the molecular work performed did, in fact, sequence the RNA genomes of the influenza viruses studied.
Now that Keller et al have managed to directly sequence RNA for the first time, the group hopes to find details of the influenza A virus’ genome that are otherwise hidden and extremely difficult to detect. Keller says this research may shed new light on the intricate lifecycle of an influenza virus as it replicates (i.e., copies) its genome and itself.
The one thing holding back this new method of direct RNA sequencing is the technology itself. According to Keller, the existing technology isn’t as accurate as it could be, and nanopore sequencers require a large amount of RNA material. Keller believes improvements in this technology will allow direct RNA sequencing to be conducted with greater accuracy and sensitivity than what is currently available.
In the meantime, this methodology opens the door on a whole new category of research impacting RNA viruses. This study, entitled “Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome” by Matthew Keller et. al., is available online from the Scientific Reports-Nature website.
Follow the link to read the full report.
Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome(SNIP)
Matthew W. Keller, Benjamin L. Rambo-Martin, Malania M. Wilson, Callie A. Ridenour, Samuel S. Shepard, Thomas J. Stark, Elizabeth B. Neuhaus, Vivien G. Dugan, David E. Wentworth &John R. Barnes
Scientific Reportsvolume 8, Article number: 14408 (2018 )
Abstract
For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods.
We designed an adapter to short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID50 6.8 × 109), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.
The primary limitations of this technology are the high read level error rate and high input material requirements. Reducing the error rate would enable multiplexing and more accurate consensus sequence determination and is a requirement for understanding nucleotide polymorphisms and genome sub-populations, particularly in viruses such as influenza that have significant intra-host diversity and or base modifications to be identified.(Continue . . . )
There are currently several bioinformatic tools for detecting DNA base modifications such as Tombo, Nanopolish, SignalAlign, and mCaller; however, RNA specific tools have yet to be released19. Currently, the RNA input requirements for direct RNA sequencing are high and are not physically achievable with most original clinical samples. While we were able to successfully sequence influenza A vRNA using much less input material than is recommended by ONT, direct sequencing of serially diluted influenza A vRNA revealed that this technique is not sensitive enough for most clinical samples and roughly four orders of magnitude less sensitive than M-RTPCR based sequencing.
Hence, direct RNA sequencing is currently limited to cultured viruses. Lessening the RNA input requirement of the direct RNA sequencing would take full advantage of the unbiased nature of direct RNA sequencing and allow for the detection and description of the rich diversity intrinsic to influenza and other viruses. The continuing effort to advance this technology by ONT will undoubtedly result in higher accuracy reads and greatly improved utility.
