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**Hogweed** · December 25, 2009, 08:37 AM

Re: Mexico releases two sequences from San Luis Potosi containing D225G and D225N

Originally posted by mixin View Post

I'm not sure about that commentary, either. The 2 from SLP are within a day of each other; but that's a highly populated area. If there was a lot of flu there, it would not be unreasonable for the researchers to find and use those 2 sequences.

I've encluded the amino acid series of letters for the D225G mutations. As you can see, there are only "B"s and "X"s; no true "G"s or "N"s; (compare it to the Catalonia with the "G" and the other Mex with a "D"). Gs's program gives all "R"s at those positiions, indicating mixed signals. I have no idea if it's accurate to say a mixed signal is an absolute G or N.

From Genbank records:

These have the mutations:
Mexico/InDRE36529 08-Aug-2009 F; age:20; Pharyngeal swab Oaxaca PKVRBQEGRMNY
Mexico/InDRE41741 14-Sep-2009 M; age:50; Pharyngeal swab Puebla PKVRBQEGRMNY
Mexico/InDRE43149 28-Sep-2009 M; age:9; Pharyngeal swab Mexico City PKVRXQEGRMNY
Mexico/InDRE50617 01-Nov-2009 M; age:40; Pharyngeal swab SLP PKVRXQEGRMNY
Mexico/InDRE50625 31-Oct-2009 M; age:25; Pharyngeal swab SLP PKVRXQEGRMNY

These do not have the mutations:
Mexico/InDRE39951 07-Sep-2009 F; age:23; Pharyngeal swab Sinaloa PKVRDQEGRMNY
Mexico/InDRE40791 20-Sep-2009 F; age:17; Pharyngeal swab Tlaxcala PKVRDQEGRMNY
Mexico/InDRE41427 23-Sep-2009 M; age:23; Lung Biopsy Mexico City PKVRDQEGRMNY
Mexico/InDRE42372 28-Sep-2009 M; age:46; Bronchoalveolar Mexico City PKVRDQEGRMNY
Mexico/InDRE43950 01-Oct-2009 M; age:59; Pharyngeal swab Mexico City KVRDQEGRMNY

This one has an actual D225G:
Catalonia/NS092145684 PKVRGQEGRMNY

Niman has replied to this elsewhere. Here's a slightly edited reply.

Characterization sheets at Genbank give the nucleotide sequence and the protein sequence for deposits. The nucleotide sequences can be quickly aligned using a number of alignment programs (i.e. ClustalW or BLAST) which are freely available at multiple sites and merely require the copy paste of the sequence (or in some cases accession number). Alignments take a few seconds and selection of appropriate displays quickly identifies mixed signals.
In the case of changes at position 225, all representations of D225G and D225N are due to mixed signals, where two signals occupy the same position. In all of the above signals the mixture is of A and G, both of which are puRines ands therefore represented by an "R" in one letter nucleotide abreviations. Thus, translation of the above sequences demonstrates that the sequences with one R either code for D225 and D225G or D225 and D225N, depending of which position is "mixed" and has an R. In the two patients who have D225G and D225N, there is an "R" at both nucleotide positions.

The rules and abreviations are standard and not subject to interpretation. However, since Genbank lists the conceptually translated protein sequence, the answer has already been generated.
For protein sequences with a mixture of wild type aspartic acid ("D") and asparagine ("N") the letter "B" is used, which again is by convention, so anyone who knows how to read a sequence will know that the "B" at position 225 means that there is a mixture of "D" and "N" (as in D225N). For the three sequences with an "X" at position 225, the actual nucleotide sequence has to be translated to see that the "R" at one position means it is a mixture of "D" and "G", while the "R" at the second position is a mixture of "D" and "N" (actaully use of both new acquistions would also produce D225S, but there has been no H1N1 sequence using both).

**sharon sanders** · December 25, 2009, 08:47 AM

Re: Mexico releases two sequences from San Luis Potosi containing D225G and D225N

Alert - As per Mixin's data these are the collection dates:

Mexico/InDRE50617 01-Nov-2009 M; age:40; Pharyngeal swab SLP PKVRXQEGRMNY
Mexico/InDRE50625 31-Oct-2009 M; age:25; Pharyngeal swab SLP PKVRXQEGRMNY

**mixin** · December 25, 2009, 09:56 AM

Re: Mexico releases two sequences from San Luis Potosi containing D225G and D225N

Hogweed,
Thanks for bringing Niman's confirmation of what I said over here. I've said many times that this isn't my field and I'm still learning. I hope he knows how much I appreciate his help. I only wish he would have addressed my question of: When there is a mixed signal of "D" and "N" is it accurate to say it's an "N"? (Or maybe he did

)

We both agree that there are mixed signals at all those positions.

Niman: For the three sequences with an "X" at position 225, the actual nucleotide sequence has to be translated to see that the "R" at one position means it is a mixture of "D" and "G", while the "R" at the second position is a mixture of "D" and "N"

I ran the ones with mutations through gs's program and posted the results in this thread:

225G Worldwide Tracking & Evaluation - FluTrackers News and Information

http://www.flutrackers.com/forum/showthread.php?t=134015&page=5

Here's one example of mixed signals at 715 and 716:

X >InDRE/50625 10-31-09 25y M Pharyngeal swab Deceased
T658A, G715R, A716R

Actually, if you look at the nucleotide portion of Mexico/InDRE50625 you can see the 2 "r"s without running it through any program.

301 tcaagccgga aatagcaata agacccaaag tgaggrrtca agaagggaga atgaactatt

**mixin** · December 26, 2009, 11:56 AM

Re: Mexico releases two sequences from San Luis Potosi containing D225G and D225N

I happened to see the following quote in study on pneumonia associated with pandemic flu; I didn't realize InDRE was in Mexico City or that SLP had its own state lab.

SLP (the state) population: 2,410,414

Samples from 37 patients were tested at UASLP, while samples from 41 patients were tested at InDRE in Mexico City or the State Public Health Laboratory in San Luis Potos?...

http://www.cdc.gov/eid/content/16/1/pdfs/09-0941.pdf (pg 7)

MexOnline.com Cities and States of Mexico - Population, Capitals, Major Cities

http://www.mexonline.com/estado.htm

**Hogweed** · December 26, 2009, 07:49 PM

Re: Mexico releases two sequences from San Luis Potosi containing D225G and D225N

Originally posted by mixin View Post

Hogweed,
Thanks for bringing Niman's confirmation of what I said over here. I've said many times that this isn't my field and I'm still learning. I hope he knows how much I appreciate his help. I only wish he would have addressed my question of: When there is a mixed signal of "D" and "N" is it accurate to say it's an "N"? (Or maybe he did

)

And Niman's reply posted elsewhere

=====

Here is a quick rundown. Since there are some fundamental misconceptions inherent in the question, I am not sure that the long version would do the trick.

Briefly, the sequences at Genbank or GISAID are called "consensus" sequences because many if not almost all samples are mixtures and there will be mixed signals generated from a sample. The mixed signals can be minimized by cloning, although such manipulations just produced a clean sequence for the clone. The clone may or may not be representative of what is the in the acual sample.

In any event, the published sequence will "erase" minor mixing, so if a position is 90% A and 10% G, it will appear as an "A" in the sequence. The actual tracing will have a major peak representing A, and a minor peak representing G at the same position.

However, when the two peaks are about the same size, indicating the ratios are close to 50/50, the sequence will have a letter signaling this mixture. Thus, an "R" at the postion indicates there is an "A" and a "G" in approximately equal proportions (R is used because A and G are both puRines). If the mixture was "C" and "T", there would be a "Y" at the position, signaling pYrimadine. All combinations have a predetermined letter.
Thus, in the examples discussed above, an R in the first position of codon 225 would indicate that D225 and D225N were present at levels that were about equal. Similarly, an R at the second position would indicate that D225 and D225G were present at similar levels.

For the cases with R at both positions, it is likely that both D225G and D225N are present (one sequence has aAT which codes for D225N, while the other has GgT which codes for D225G). Since both are present, a mixed signal would be generated at both positions (represented by tandem R's in the nucleotide sequence).

However, it is possible (but highly unlikely) that the tandem R's are created by one sequence that is wild type (GAT), while the second sequence has two changes (agT) which would create D225S (and neither D225G nor D225N would be present). However, since D225G and D225N have been seen multiple times each (including the other thee patients from Mexico), while D225S has not been reported, it is likely that the tandem R's are due to aAT in one sequence, and GgT in the other.

**mixin** · December 27, 2009, 07:13 AM

Re: Mexico releases two sequences from San Luis Potosi containing D225G and D225N

Hogweed,
Thanks again.

Niman: Since there are some fundamental misconceptions inherent in the question, I am not sure that the long version would do the trick.

Since we both seem to be saying the same thing, I'm not sure what my fundamental misconceptions are. Maybe we are just miscommunicating; I'll try again.

I'm only going to talk about position 715 (the first positon in the codon) so no one gets even more confused.

Nucleotide: agg gat caa, Amino acid: PKVRDQEGRMNY = D225
Nucleotide: agg aat caa, Amino acid: PKVRNQEGRMNY = D225N
Nucleotide: agg rat caa, Amino acid: PKVRBQEGRMNY Is it accurate to also call this D225N?

Just a straight "Yes" or "No" will be sufficient.

**mixin** · December 30, 2009, 05:45 AM

Re: Mexico releases two sequences from San Luis Potosi containing D225G and D225N

Gs updated one of his files and it contains 3581 human and swine H1s from genbank except for pandemic H1N1. I was curious to see how rare these mutations are, so I searched his records and found the following:

715 has 94 total; 88 have "A", 6 have "R" = D225N
716 has 284 total; 274 have "G" and 10 have "R" = D225G

There are a lot more than I thought. About 1/3 of the 715 mutations are found in the Memphis 1983 human sequences.

D225G seems to be pretty scattered in time and place. There are quite a few 1976/1977 Tennessee pig sequences that have it, then Memphis human sequences from 1978 have it. Wisconsin pigs had it in 1997/1998.

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Mexico releases two sequences from San Luis Potosi containing D225G and D225N

Mexico releases two sequences from San Luis Potosi containing D225G and D225N

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