<p>Is "user5054" the same as "user5504"? No? Exactly. Not only does order matter, it's incredibly vitally important. <code>AG</code> and <code>GA</code> are completely and totally different from each other. If this is in a coding region, then the resulting amino acid is undoubtedly changed (fun fact, the <a href="https://en.wikipedia.org/wiki/DNA_codon_table" rel="nofollow noreferrer">only exception</a> is <code>TAG</code> and <code>TGA</code>). If it's at a <a href="https://en.wikipedia.org/wiki/RNA_splicing" rel="nofollow noreferrer">splice site</a> then splicing is likely altered (AG is part of a splice acceptor site). If it's at the binding site of something then it wouldn't be surprised if it didn't bind any more.</p>

CC BY-SA 3.0
2017-08-22T07:36:00.350Z
I think "user5054" vs. "user5504" example is misleading. My example would be that the sets {1,2} or {2,1} mean exactly the same thing, while if those represent permutations, they are different. So it depends on the case. In the SNP case, each of 'G' or 'A' in 'GA' or 'AG' are the nucleotides in the one of the two strands of DNA, right? So, both 'GA' and 'AG' are heterozygous alleles for the same SNP.Then what I understand is that the ordering of the strands matter,because maybe the transcribed strand's nucleotide is written first while representing the SNP?I think your reply needs more detail.
user5054
CC BY-SA 3.0
2017-08-22T07:50:49.283Z
Is this actually a SNP or is it an InDel? If it's a SNP then you're correct that it doesn't matter (assuming no phasing information). My presumption is that this is not a SNP, but rather an InDel.
Devon Ryan
CC BY-SA 3.0
2017-08-22T21:20:31.730Z
Yeah, this is a SNP analysis. I should have been clearer when I asked the question. Please see my comment to Emily_Ensembl above. The statement in my previous comment to your reply saying "...the ordering of the strands matter, because maybe the transcribed strand's nucleotide is written first while representing the SNP?" was completely out of confusion due to being new to genetic analyses. Apparently, Each of G or A in "GA" or "AG" do not refer to different strands of DNA, but refer to the different chromosomes of an individual.
user5054
CC BY-SA 3.0
2017-08-22T06:40:57.690Z
2017-08-22T06:40:57.690Z
Devon Ryan
<p>Most association analyses are carried out at a single SNP level, so AG and GA are likely to indicate a heterozygous genotype at a particular location. However, the precise notation matters.</p>

<p>As @Emily_Ensembl has alluded to, for VCF files, A/G indicates unphased SNPs (order unknown, and shouldn't be considered in analyses), whereas A|G indicates phased SNPs (order matters).</p>

<p>It is possible to carry out association analyses by combining multiple variants into a single haplotype, in which case the order would also matter. It's unlikely that this is the case, because such haplotype variant analyses are substantially more complicated to carry out, and will typically be paired with information about SNP linkage (statistic D´ or r²).</p>

CC BY-SA 3.0
2017-08-22T09:19:27.233Z
2017-08-22T09:19:27.233Z
gringer
<p>Could you please show us the context in which this appears, as you seem to be interpreting this differently to Devon.</p>

<p>If it's appearing as you say, GA and AG, then Devon is right, this usually means that the sequence goes ###AG### or ###GA###, which are two very different sequences.</p>

<p>If, however, as you're implying, the sequences are actually ###G### or ###A###, then semantically, the two ways of writing it have the same outcome. Normally, however, this is written as A/G or G/A.</p>

<p>Alternatively, you're looking at genotypes, which would usually appear as A|G and G|A. In this case, both individuals are heterozygotes, and in terms of the individual SNP they are the same. However the side of the pipe is relevant as this tells you which homologous chromosome it is on. This allows you to calculate LD with respect to neighbouring SNPs, and to determine the actual sequences of the proteins in that individual.</p>

CC BY-SA 3.0
2017-08-22T21:15:08.437Z
Thanks for the reply. I am looking at genotypes. I realized that I had a bug while retrieving the genotypes, so what plink thinks as being different was not in fact GA vs. AG (each of which would really refer to heterozygous individuals as I replied to Devon), but it was GA and AA, where one is homozygous and the other is heterozygous. So, it is totally normal that those alleles are different for SNP association analysis.
user5054
CC BY-SA 3.0
2017-08-22T21:55:27.297Z
"However the side of the pipe is relevant as this tells you which homologous chromosome it is on. This allows you to calculate LD with respect to neighbouring SNPs, and to determine the actual sequences of the proteins in that individual." -- Could you please explain this a little further?
user5054
CC BY-SA 3.0
2017-08-23T07:39:57.800Z
If you have two variants, separated by, say 50, bp and they both fall in one gene. The genotype in one individual is G|A for the first variant and C|A for the second. That means that one of their genes contains a G and a C, the other gene contains an A and an A. 

This might be relevant in various ways. In a population study, you might just be generally interested to know whether G...C and A...A are more common than G...A and A...C. This would also matter in a phenotype association study, as only one may be causative, but if they always turn up together then how would you know which?
Emily_Ensembl
CC BY-SA 3.0
2017-08-23T07:45:21.253Z
(ran out of space!) It could also be that the phenotype only occurs when both variants occur, or is perhaps alleviated by the pairing. If both those variants affect amino acids, and those two amino acids are in contact, mutating one only could disrupt the connection, but mutating both the connection is maintained. Or if they're functionally redundant amino acids, one is able to compensate for the other, so when only one occurs, there's no phenotype, but when both do there is a phenotype.
Emily_Ensembl
CC BY-SA 3.0
2017-08-23T07:46:35.677Z
There's a pair of courses here on introduction to genetic variation, which explains a lot about things like LD, effect on proteins etc: http://www.ebi.ac.uk/training/online/course/human-genetic-variation-i-introduction http://www.ebi.ac.uk/training/online/course/human-genetic-variation-ii-exploring-publicly-available-data
Emily_Ensembl
CC BY-SA 3.0
2017-08-22T07:53:00.347Z
2017-08-22T07:53:00.347Z
Emily_Ensembl
<p>I am using plink to check the association between a phenotype and the SNPs of a gene. plink says the phenotype is significantly associated with a SNP on that gene, and when I check the SNP alleles, for 6 out of 49 samples, the nucleotides are 'GA', and for the rest, they are 'AG', so only the order is different. I am not a biologist, but my basic genetics knowledge says that those two are semantically the same. However, obviously, plink does not agree with me and it associates this SNP with a small p-value since 'GA' samples have a higher value for the phenotype than 'AG' samples. I am very new to SNP analysis, any help is appreciated.</p>

<snp><gwas><plink>
Is there any difference between SNPs 'AG' and 'GA' in association analyses?
# Is there any difference between SNPs 'AG' and 'GA' in association analyses?

(Asked by: username_0 on 2017-08-22)

I am using plink to check the association between a phenotype and the SNPs of a gene. plink says the phenotype is significantly associated with a SNP on that gene, and when I check the SNP alleles, for 6 out of 49 samples, the nucleotides are 'GA', and for the rest, they are 'AG', so only the order is different. I am not a biologist, but my basic genetics knowledge says that those two are semantically the same. However, obviously, plink does not agree with me and it associates this SNP with a small p-value since 'GA' samples have a higher value for the phenotype than 'AG' samples. I am very new to SNP analysis, any help is appreciated.

**Tags:** snp, gwas, plink

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## 3 answers

### Answer by username_3 on 2017-08-22. Score: 8 | Accepted answer

Could you please show us the context in which this appears, as you seem to be interpreting this differently to Devon.

If it's appearing as you say, GA and AG, then Devon is right, this usually means that the sequence goes ###AG### or ###GA###, which are two very different sequences.

If, however, as you're implying, the sequences are actually ###G### or ###A###, then semantically, the two ways of writing it have the same outcome. Normally, however, this is written as A/G or G/A.

Alternatively, you're looking at genotypes, which would usually appear as A|G and G|A. In this case, both individuals are heterozygotes, and in terms of the individual SNP they are the same. However the side of the pipe is relevant as this tells you which homologous chromosome it is on. This allows you to calculate LD with respect to neighbouring SNPs, and to determine the actual sequences of the proteins in that individual.

> **Comments:**
> **username_0**: Thanks for the reply. I am looking at genotypes. I realized that I had a bug while retrieving the genotypes, so what plink thinks as being different was not in fact GA vs. AG (each of which would really refer to heterozygous individuals as I replied to Devon), but it was GA and AA, where one is homozygous and the other is heterozygous. So, it is totally normal that those alleles are different for SNP association analysis.  
> **username_0**: "However the side of the pipe is relevant as this tells you which homologous chromosome it is on. This allows you to calculate LD with respect to neighbouring SNPs, and to determine the actual sequences of the proteins in that individual." -- Could you please explain this a little further?  
> **username_3**: If you have two variants, separated by, say 50, bp and they both fall in one gene. The genotype in one individual is G|A for the first variant and C|A for the second. That means that one of their genes contains a G and a C, the other gene contains an A and an A.   This might be relevant in various ways. In a population study, you might just be generally interested to know whether G...C and A...A are more common than G...A and A...C. This would also matter in a phenotype association study, as only one may be causative, but if they always turn up together then how would you know which?  
> **username_3**: (ran out of space!) It could also be that the phenotype only occurs when both variants occur, or is perhaps alleviated by the pairing. If both those variants affect amino acids, and those two amino acids are in contact, mutating one only could disrupt the connection, but mutating both the connection is maintained. Or if they're functionally redundant amino acids, one is able to compensate for the other, so when only one occurs, there's no phenotype, but when both do there is a phenotype.  
> **username_3**: There's a pair of courses here on introduction to genetic variation, which explains a lot about things like LD, effect on proteins etc: http://www.ebi.ac.uk/training/online/course/human-genetic-variation-i-introduction http://www.ebi.ac.uk/training/online/course/human-genetic-variation-ii-exploring-publicly-available-data  

---

### Answer by username_1 on 2017-08-22. Score: 4

Is "user5054" the same as "user5504"? No? Exactly. Not only does order matter, it's incredibly vitally important. `AG` and `GA` are completely and totally different from each other. If this is in a coding region, then the resulting amino acid is undoubtedly changed (fun fact, the [only exception](https://en.wikipedia.org/wiki/DNA_codon_table) is `TAG` and `TGA`). If it's at a [splice site](https://en.wikipedia.org/wiki/RNA_splicing) then splicing is likely altered (AG is part of a splice acceptor site). If it's at the binding site of something then it wouldn't be surprised if it didn't bind any more.

> **Comments:**
> **username_0**: I think "user5054" vs. "user5504" example is misleading. My example would be that the sets {1,2} or {2,1} mean exactly the same thing, while if those represent permutations, they are different. So it depends on the case. In the SNP case, each of 'G' or 'A' in 'GA' or 'AG' are the nucleotides in the one of the two strands of DNA, right? So, both 'GA' and 'AG' are heterozygous alleles for the same SNP.Then what I understand is that the ordering of the strands matter,because maybe the transcribed strand's nucleotide is written first while representing the SNP?I think your reply needs more detail.  
> **username_1**: Is this actually a SNP or is it an InDel? If it's a SNP then you're correct that it doesn't matter (assuming no phasing information). My presumption is that this is not a SNP, but rather an InDel.  
> **username_0**: Yeah, this is a SNP analysis. I should have been clearer when I asked the question. Please see my comment to Emily_Ensembl above. The statement in my previous comment to your reply saying "...the ordering of the strands matter, because maybe the transcribed strand's nucleotide is written first while representing the SNP?" was completely out of confusion due to being new to genetic analyses. Apparently, Each of G or A in "GA" or "AG" do not refer to different strands of DNA, but refer to the different chromosomes of an individual.  

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### Answer by username_2 on 2017-08-22. Score: 3

Most association analyses are carried out at a single SNP level, so AG and GA are likely to indicate a heterozygous genotype at a particular location. However, the precise notation matters.

As @Emily\_Ensembl has alluded to, for VCF files, A/G indicates unphased SNPs (order unknown, and shouldn't be considered in analyses), whereas A|G indicates phased SNPs (order matters).

It is possible to carry out association analyses by combining multiple variants into a single haplotype, in which case the order would also matter. It's unlikely that this is the case, because such haplotype variant analyses are substantially more complicated to carry out, and will typically be paired with information about SNP linkage (statistic D´ or r²).


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