Hi Kimberly, are you purifying your pcr amplicons before cloning?, it looks like your are cloning unspecific amplicons out of your pcr. How do your bands look like in an agarose gel? for some reason small fragments clone better than bigger ones, then if you have unspecific small fragments in your reaction those are gonna be the ones cloned instead of the one that you are interested in.

I am gel purifying my band prior to sequencing and/or cloning.

Then I would take care of the amount of product (RNA, cDNA, even PCRs) that I´d be handling. Just to see if technics such as RNA extraction, RTs, etc, are being done with the amount of material that is required. I do this for Influenza A virus and it works fine!

Most RNA virus have secondary structure, which is the reason to cause problem during cDNA sequencing. I had exactly same problems when I tried to sequence LCMV genome. As a matter of fact, even the recombinant plasmid DNA carrying LCMV cDNA is not very stable and tend to change during propagation. If you try to use small piece cloning strategy, try to use a true stable RecA- bacterial strain, I prefer HB101 and culture for short time (less than 12 hours). Otherwise, sequence direct PCR fragment is not a bad approach. Bear in mind that Trisol based RNA approach is not a viable method for viral RNA extraction. I use to use Protease K digest +LiCl precipitation of RNA. Later my colleague suggest that Qiagen RNA kit is also a viable method for sequencing purpose. We used it for LCMV as well as influenza A virus sequencing.

Hi Kimberly,

We have done this for human metapneumovirus, multiple strains. We amplified genome fragments (~2-3kb) by one step RT-PCR, then TA cloned into pGEM. We gel purify RT-PCR product prior to cloning. More expensive than direct sequencing of PCR products, but we found that direct sequencing of the PCR product didn't yield high-quality sequences, even after purification. I think it's related to the amount of product after purification, and the fact that even with purification there may be some contaminating primers. Good luck,

john

Follow influenza virus genome seq. Literature.

Hi Kimberly,

Normally I am sequencing viral genome ss RNA viruses directly with or without purification from the PCR products, and it goes always well.

You may check all the procedures you are conducting starting from the RNA Prep. until samples seq. Check the effeciency of each step. For RNA pre. I am using the protocol described by Boom et al. 1999. For the PCR I am using usually 4x or mater mix even more volume to ensure having big amount of cDNA. its always better if possible to use the viral isolates ....

Good luck

Are you cloning on the same day as the final PCR step? We've found that cloning of large amplicons depends on "fresh" ends, and that it's best to clone immediately after the PCR; otherwise, a short incubation with fresh Taq and dATP can make a big difference.

I would advise against subcloning and then sequencing fragments. Your virus population is most likely not clonal (unless you work with plaque-purified viruses, or a reverse genetics system), so that you have many quasispecies with slightly different genomes. If you subclone fragments, you get sequence information of one random member of the whole pool, whereas if you directly sequence from PCR products, you should get a majority sequence.

I routinely sequence EBOV viruses (NNSV with 19 kB genome), and this works quite well. I have designed the PCR primers in a way that all my fragments are about 700 bp (with about 100 bp overlaps between fragments), with primers having the same Tms, do the RT with Superscript III and the PCR with IProof. I can send you a detailed protocol if you need it.

For the reaction to work well try to have a lot of viral RNA, I have also found primer directed RT as a first stand-alone step (Sigma-Aldrich cDNA kit) with 1 hr reactions is better than a one-step RT-PCR rxn. Followed this with jump start accuTaq (Sigma) works the best for large cDNAs ( I have cloned ~ 6 kb genomes. With pfx or "pfusion all I get is small fragments. Jump start has some Taq which means TA-topo will be needed for cloning. If your RNA has alot of secondary structure you may need to add some DMSO or betaine to give you a lower Tm.

Good luck with your research

Hi Kimberley, I'm not sure if you are aware of it, but the Virology group on Linked In has about 1400 virology experts, I'm sure someone there will also be able to help you, although it seems as you have lots of avenues to follow here too. Good luck.

We have done a considerable amount of sequencing of negative strand RNA viruses

The first approach provided the complete genome sequence for several novirhabdoviruses and vesiculoviruses of approximately 11,000 nt in length. The genome was divided into overlapping fragments of 500-600 nucleotides and these regions were amplified using conventional RT-PCR using RNA extracted from PEG precipitated (concentrated ) virus. In our hands the amplification of larger fragments, irrespective of the reverse transcriptase, is very inefficient and we always favour the shorter amplification products. In your case this may require 40 primer sets, but the amplifications can be easily achieved in duplicate on a single 96 well plate and the products sequenced directly using a single plate for the forward reactions and a second plate for the reverse reactions. The whole process can be done in a week or so. This process avoids any cloning issues.

The second approach we have used is 454 de novo sequencing using RNA from vesiculoviruses concentrated using a PEG precipitation in the same way. I can provide full details of the amplifications methods used up to the point of sending the sample for commercial sequencing if you wish. A 16th of a 454 sequencing plate was sufficient to obtain the complete genome, excluding the 3’ and 5’ termini sequences which were generated using standard 3’ and 5’ RACE procedures.

Finally, we are currently in the process of sequencing multiple genomes on a single plate using the equivalent of MID tagged primers, but we are still waiting for the data on this.

Excuse me to add a question here, but I got some doubt if I can use the same primers used in the RT PCR to directly sequencing the PCR product. I would like to thank all the considerations about this matter.