Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

The overall goal of this procedure is to prepare sequencing libraries of RNA viruses directly from clinical samples to enable viral surveillance and evolutionary studies. This method can answer key questions in the fields of viral genomics and evolution, such as how RNA viruses mutate within and between hosts and how they spread within a population. The main advantage of this technique is that it's unbiased.

It requires no prior knowledge of the viral sequence for library preparation and can be used to sequence any virus. The implications of this technique extend towards detecting and combating viral diseases like Lassa fever, because the diagnostics and the therapeutics we develop rely fundamentally on the genome sequence of the virus. Demonstrating selective depletion will be Adrianne Gladden.

Demonstrating CDNA synthesis will be Sarah Winnicki. And demonstrating DNA library construction will be Dolo Nosamiefan. To begin, prepare 5X hybridization and 10X RNase H reaction buffers in nuclease-free water with linear acrylamide carrier as described in table one of the text protocol.

In a 96 well PCR plate in a metal block on ice, set up the hybridization reaction by combining previously extracted and DNase treated viral RNA and ribosomal RNA depletion oligos and oligo(dT)s. Note all reactions in this protocol are prepared and distributed into strip tubes for use with a multi channel pipette. Vortex the reaction gently and thoroughly, then centrifuge at 280 times g and room temperature for one minute.

Incubate at 95 degrees Celsius for two minutes, slowly ramping down to 45 degrees Celsius at minus 0.1 degree Celsius per second. Then pause the thermocycler at 45 degrees Celsius. Set up an RNase H reaction mix in a metal block on ice, then preheat the solution at 45 degrees Celsius for two minutes.

With the 96 well plate still in the thermocycler at 45 degrees Celsius, add the preheated RNase H mix to the hybridization reaction. Mix well by gently pipetting six to eight times, then incubate at 45 degrees Celsius for another 30 minutes before placing on ice. Next, set up the DNase reaction mix in a metal block on ice as described in table one of the text protocol.

Add the mix to the RNase H reaction in the plate. Vortex gently and thoroughly, then centrifuge at 280 times g and room temperature for one minute. Incubate the plate at 37 degrees Celsius for 30 minutes, then add five microliters of 0.5 molar EDTA to stop the reaction.

After gently and thoroughly vortexing the plate, centrifuge at 280 times g and room temperature for one minute. Use a 1.8X volume of RNA beads to clean up the reaction per the manufacturer's suggestion, and elute in 11 microliters of nuclease-free water. Store depleted RNA samples at minus 80 Celsius overnight.

Special care should be taken at this step and sample loss or degradation could occur. To carry out CDNA synthesis, in a 96 well PCR plate in a metal block on ice mix ribosomal RNA carrier depleted RNA with random primers according to the text protocol. After gently vortexing and spinning the plate, in a thermocycler heat the mixture to 70 degrees Celsius for ten minutes.

Immediately after heat denaturation, place the RNA in a metal block on ice for one to five minutes. Next, after preparing the first strand synthesis reaction mix, add it to the RNA random primer mix in the plate. Then, following the vortex and centrifuge, incubate at 22 to 25 degrees Celsius for ten minutes.

Incubate the plate at 55 degrees Celsius in an air incubator for 60 minutes. Then place the plate in a metal block on ice to terminate the reaction. Now, set up the second strand synthesis reaction mix and add it to the first strand synthesis reaction in the plate.

After vortexing and spinning as before, incubate the reaction at 16 degrees Celsius for two hours. Place the plate in a metal block on ice then inactivate the reaction by adding five microliters of 0.5 molar EDTA before mixing and spinning as before. With a 1.8X volume of DNA beads, clean up the reaction and use nine microliters of elution buffer or EB to elute the samples.

Save one microliter for quantification. For safe cold storage, store double stranded CDNA at 4 degrees Celsius overnight or minus 20 degrees Celsius for long term storage. To construct a DNA library, transfer four microliters of CDNA to a 96 well plate and save the remaining CDNA for a second attempt at detection if needed.

While working in a metal block on ice, set up the tagmentation reaction as described in table one of the text protocol. Then add the mix to the CDNA in the 96 well plate. After vortexing and spinning, incubate the plate at 55 degrees Celsius for five minutes then hold it at 10 degrees Celsius.

Once at 10 degrees Celsius, immediately add 2.5 microliters of neutralized tagment buffer to end the reaction. After centrifuging the plate at 280 times g and room temperature for one minute, incubate at room temperature for five minutes. Now set up the PCR amplification reaction in a metal block on ice as described in text protocol.

After vortexing and spinning the plate, carry out PCR. To prepare the library, use EB to bring up the samples to 50 microliters. Using a 0.6X volume of DNA beads, clean up the reaction and elute in 15 microliters of EB.Pool the libraries at the lowest molar concentration of one nanomolar or greater.

If the library is below one nanomolar, add a small volume of library to the pool. Finally, use a 0.7X volume of DNA beads to clean up the pool as outlined earlier in the video, then use 15 microliters of EB to elute the samples. Analyze according to the text protocol.

As shown here, the protocol described in this video enriched unique Lassa virus content at least fivefold in all samples with at least one million copies of 18S ribosomal RNA. This graph illustrates the depletion of poly(rA)carrier reduced homopolymer sequences of A and T in libraries, results in cleaner preparations and ensures better quality sequencing reads. Finally, this figure demonstrates that final libraries from low input viral clinical samples often have a broad fragment plane from 150 to 1000 base pairs.

Once mastered, this technique can be done in three days if performed properly. While attempting this procedure, it's important to remember that most clinical samples have very little nucleic acid material. Therefore sample loss, contamination and RNA degradation are common.

Following this procedure, other methods like RNA seek can be performed to answer additional questions like how does the host respond to viral infection. After its development, this technique paved the way for researchers in the field of viral surveillance to trace transmission paths of viruses like Ebola and Lassa, and other circulating pathogens in West Africa. After watching this video, you should have a good understanding of how to prepare sequencing libraries of RNA viruses directly from clinical samples, enabling viral surveillance and evolutionary studies.

Don't forget that working with viral pathogens can be extremely hazardous. Precautions such as using the proper personal protective equipment and biosafety level regulations should always be taken when performing this procedure.