Great! This is exactly what I was looking for.
For the sake of clarity, maybe we should focus on the Canadian pigs, since they are a very dramatic example, and you seem to have had some contact with Olsen already.
I would like to ask a number of questions:
1. Did their lab have the strains with long regions of sequence identity with the Canadian viruses, like Tennessee/24/77, Tennessee/26/77, NC/98, Korea/CY/02?
2. How likely is it that one of these strains could have contaminated the Canadian samples? What steps are taken to prevent this kind of contamination, and how is their effectiveness assessed?
3. The sequencing process. There is a series of steps: culturing, RNA extraction, reverse transcription, PCR and actual sequencing.
a. Are different segments sequenced from different tubes?
b. What primers are used for the PCR? Are they expected to amplify a whole segment, or do different primers at different ends of a segment amplify overlapping chunks of sequence?
c. How is the sequencing done: is the whole sequence obtained in one step, or are overlapping fragments put together to give a sequence for each segment? Would this be different for the longer and shorter segments?
4. If there is contamination, so that RNA's from two strains are present in the same tube, could this result in a sequence that looks like a recombinant? Would it make sense that the breaks occur at particular places (because of the PCR primers / sequencing fragments / some other reason?)
5. Would they be willing/able to check if contamination may have occurred, and/or resequence the isolates?
You can contact the lab directly or go through Genbank -- whichever is the best way to get an answer! Also, it would be great to forward this to the lab people on the board, and get their input.
I greatly appreciate this -- you've been very generous with your time and attention.
