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A question about recombination (could contamination be an issue?)
Re: A question about recombination (could contamination be an issue?)
the Wisconsin-DQ and the Memphis/104 sequences could
have been generated in a lab, but the Canadian swine were not.
There was a paper by Olsen et.al. describing, how they
were collected on different farms and different times.
They had multiple recombinations and identities of long subsequences,
5-10 different viruses with intermediates
I think I wasn't being very clear about what I mean by "contamination". I don't necessarily mean that the actual host is infected by different viruses (though of course that's also possible). What I mean is that the contamination might occur in the lab itself, after the samples have been isolated. So the samples could be from different places, times etc. Once they're brought into the lab they have to be cultured (in some cell line, or chicken eggs) before they're sequenced. I'm thinking that if the lab technician is sloppy, viruses from strain A could fly around and infect a culture that was supposed to contain strain B.
So the key piece of evidence that this is not the case would be not samples from different times or places, but submitted by different labs. I think most of the Canadian swine examples are from the same lab, though I could be wrong.
Does this make sense?
Although all of the Canadian sequences came from the same lab, there are two different factors that would pretty much eliminate contamination. One is the number of different viruses involved. The two best examples are PB2 and PA which involved 1977 Tennessee sequences. However, the Tennesee sequences are from two different isolates (numbers 24 and 26).
Moreover, the two Tennessee sequences are not in the other gene segments to any appreciable degree. Moreover, within the two genes above there are 1998, 2002, and 1931 sequences, so the number of contaminating viruses would be large. Again, these sources are predominantly in PB2 and PA.
In addition, the PB1 sequence is human, but from the mid 90's (even though the swine isolates are from 2003 and 2004)
and the PB1 doesn't have any long stretches of swine sequences (all of the sequences listed above are swine). Moreover, two of the isolates have human H1 and N1, which again do not have "contaminating" swine sequences.
Thus, the multiple recombination events ares quite real. It is in "slow motion", so the long stretches of identity persist, some as long as more than 70 years, showing that the polymerase can copy sequences with a high degreee of fidelity.
Although all of the Canadian sequences came from the same lab, there are two different factors that would pretty much eliminate contamination. One is the number of different viruses involved. The two best examples are PB2 and PA which involved 1977 Tennessee sequences. However, the Tennesee sequences are from two different isolates (numbers 24 and 26).
Let's consider the Canadian PB2 alignments. I think they break roughly into 3 groups. The first group is TN/24/77, 48235 and 55383. The alignment has the form
TN A A A
48235 B A B
55383 B A B
the breaks are around nt. 220 and nt. 1930
The second group is NC/98, 23866 and 11112. The alignment looks like
NC A A ?
23866 B B B
11112 B A B
the breaks are at 730 and 1570
The third group is Korea/02, 57561, 56626, 53518. It looks roughly like
Korea A A A
57561 B B B
56626 C B C
53518 C A A
the breaks are at 560 and 1590
I find this last alignment very puzzling. It seems like there are separate recombination events: 56626 got a piece of 57561, and 53518 got a piece of Korea/02. But why do they occur at the exact same place in the sequence?? This doesn't seem to make sense.
The other thing that's puzzling about all three alignments is that there seems to be a pattern where there are always 2 recombination events that occur at roughly complementary positions with respect to each other on the segment. How can this be explained?
It seems hard to believe that there would be three separate contamination events in one lab. But I guess if the lab is a complete mess it's not impossible.
Something that would settle this is simply resequencing the samples. If there is a contamination, one would expect that the resequenced sequences would be very different (a new pattern of RT jumping). But if they're true recombinants, one would expect the same sequence upon resequencing. Is anyone aware whether any of the labs that reported such dramatic cases of apparent recombination has attempted to resequence the strains? Did Olsen try to do that? People in the field are inclined to disbelieve recombination, for whatever reason, so reproducibility is key ("extraordinary claims require extraordinary proof", etc.)
Let's consider the Canadian PB2 alignments. I think they break roughly into 3 groups. The first group is TN/24/77, 48235 and 55383. The alignment has the form
TN A A A
48235 B A B
55383 B A B
the breaks are around nt. 220 and nt. 1930
The second group is NC/98, 23866 and 11112. The alignment looks like
NC A A ?
23866 B B B
11112 B A B
the breaks are at 730 and 1570
The third group is Korea/02, 57561, 56626, 53518. It looks roughly like
Korea A A A
57561 B B B
56626 C B C
53518 C A A
the breaks are at 560 and 1590
I find this last alignment very puzzling. It seems like there are separate recombination events: 56626 got a piece of 57561, and 53518 got a piece of Korea/02. But why do they occur at the exact same place in the sequence?? This doesn't seem to make sense.
The other thing that's puzzling about all three alignments is that there seems to be a pattern where there are always 2 recombination events that occur at roughly complementary positions with respect to each other on the segment. How can this be explained?
It seems hard to believe that there would be three separate contamination events in one lab. But I guess if the lab is a complete mess it's not impossible.
Something that would settle this is simply resequencing the samples. If there is a contamination, one would expect that the resequenced sequences would be very different (a new pattern of RT jumping). But if they're true recombinants, one would expect the same sequence upon resequencing. Is anyone aware whether any of the labs that reported such dramatic cases of apparent recombination has attempted to resequence the strains? Did Olsen try to do that? People in the field are inclined to disbelieve recombination, for whatever reason, so reproducibility is key ("extraordinary claims require extraordinary proof", etc.)
Some of the recombination patterns are due to nested segements. In other cases, more than one isolate has the same origin,
For resequencing, Beijing Genone Institute did that.
They submitted a number of H5N1 sequences with obvious recombination. A year later they resequenced and the results were identical.
You are barking up the wrong tree. The recombination is quite real and not due to lab artifact. The reasons the data from the Canadian swine are real have been explained. Trying to argue lab artifact for that many changes really is not very productive. No one is making that argument for the Canadian swine data (which would require 50-100 lab errors to generate the data for the eight gene segments).
They submitted a number of H5N1 sequences with obvious recombination. A year later they resequenced and the results were identical.
You are barking up the wrong tree. The recombination is quite real and not due to lab artifact. The reasons the data from the Canadian swine are real have been explained. Trying to argue lab artifact for that many changes really is not very productive. No one is making that argument for the Canadian swine data (which would require 50-100 lab errors to generate the data for the eight gene segments).
Thanks for the link. Which sequences were resequenced? Following this thread:
You say that possibly henan/wu/2004 was a resequencing of henan/210/2004.
Am I missing what the others are?
For what it's worth, my belief is that the recombination is real. The evidence is very impressive. I was simply trying to understand why the Influenza field seems to be ignoring it. Lab contamination is the best objection I could come up with. I'm just looking for the most definitive way to refute it.
I'm curious -- if no one is making the argument of lab artifact, what could they possibly be saying? How do they argue against recombination?
You say that possibly henan/wu/2004 was a resequencing of henan/210/2004.
Am I missing what the others are?
For what it's worth, my belief is that the recombination is real. The evidence is very impressive. I was simply trying to understand why the Influenza field seems to be ignoring it. Lab contamination is the best objection I could come up with. I'm just looking for the most definitive way to refute it.
I'm curious -- if no one is making the argument of lab artifact, what could they possibly be saying? How do they argue against recombination?
All of the original sequences were resequenced, but the one you cited above has the most obvious recombination among the early set (there were many more in the expanded later set).
The denial in the influenza community is in three stages.
1. It can't be true.
2. Even if it is true, it can't be important.
3. We knew it all along.
Most of those in category 1 haven't looked at the data. Many of those who have looked at the data are in category 2. All very small number are in category 3, but that will change,
Re: A question about recombination (could contamination be an issue?)
I'm not so fast...
Also internet problems today.
So, here are the Wisconsin pictures,
looks like there could be recombination in PB1 too ?
Also many changes and clustered mutations, 90% 3rd base in PB2.
The full sequences were submitted last year, this could mean
that there will be a paper in not too distant future about this ?!?
So, here are the Wisconsin pictures,
looks like there could be recombination in PB1 too ?
Also many changes and clustered mutations, 90% 3rd base in PB2.
The full sequences were submitted last year, this could mean
that there will be a paper in not too distant future about this ?!?
Actually, there is a paper associated with the DQ turkey:
Virology. 2005 Sep 15;340(1):70-83.
Evolution of H9N2 influenza viruses from domestic poultry in Mainland China.
Li C, Yu K, Tian G, Yu D, Liu L, Jing B, Ping J, Chen H.
They submitted the sequences of a bunch of H9N2 viruses and the DQ turkey/Wisconsin/66. It's not a new lab-generated sequence. In fact, the CY turkey sequence was submitted later -- in the big 2006 Webster Science paper.
In this particular case, lab contamination seems to be the most economical explanation. Note that there is a sequence from the same submission -- chicken/Heilongjiang/35/00 -- whose NP is almost identical to the NP of turkey/Wisconsin/66, despite being 34 years later. Both that and the apparent recombination between identical strains could be explained by contamination.
All of teh original sequences were resequenced, but the one you cited above has the most obvious recombination among the early set (there were many more in the expanded later set).
I'm probably missing something here. I still can't find which sequences were resequenced. Looking at the NCBI database, I don't see any of these Chinese H5N1 sequences that have the same name (like chicken/Henan/wu/2004 etc.) but different accession numbers. Can you give a specific example where the same strain was resequenced, and how you can tell that it is the same strain being resequenced?
I'm probably missing something here. I still can't find which sequences were resequenced. Looking at the NCBI database, I don't see any of these Chinese H5N1 sequences that have the same name (like chicken/Henan/wu/2004 etc.) but different accession numbers. Can you give a specific example where the same strain was resequenced, and how you can tell that it is the same strain being resequenced?
The names changed. One pair was described in the commentary. For most of the genes the sequences are identical. For one or more the difference is 1 BP.
I'm probably missing something here. I still can't find which sequences were resequenced. Looking at the NCBI database, I don't see any of these Chinese H5N1 sequences that have the same name (like chicken/Henan/wu/2004 etc.) but different accession numbers. Can you give a specific example where the same strain was resequenced, and how you can tell that it is the same strain being resequenced?
If you look at the sequence and the sequencers, it is pretty obvious. I'm not sure why you seem to ask the same questions again and again. The answer was explained in the commentary, as well as my earlier comments on this thread.
The story is in the sequence and it is VERY obvious.
The names changed. One pair was described in the commentary. For most of the genes the sequences are identical. For one or more the difference is 1 BP.
Hmm... so do you know that both sequences refer to the same sample, or are you just assuming it based on sequence identity?
I don't mean to harp on this, I'm just wondering if there is a way to tell from the database that something has been resequenced, other than guessing.
Please. If you don't want to look at the sequence, check out the definition of OBVIOUS (and guessing).
OK. How is it obvious?
There are endless examples of definitely different samples, often submitted by the same lab, that are nearly identical in sequence. Obviously, strains circulating at the same time in the same place tend to be very close to each other. That's not the same thing as resequencing the same physical sample.
There are endless examples of definitely different samples, often submitted by the same lab, that are nearly identical in sequence. Obviously, strains circulating at the same time in the same place tend to be very close to each other. That's not the same thing as resequencing the same physical sample.
Please. All 8 gene segments have been submitted twice. The identity is OBVIOUS.
You have been told how to check the OBVIOUSNESS, yet instead of looking at the data, you continue to post comments that have little relevance. The sequences were resubmitted and the resubmissions are OBVIOUS.
Your comments have moved well into the annoying category.
If you want to argue the data, I suggest you look at the data. You should be able to quickly see that there is no argument.
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