Switch or leave as separate linkage groups?

[Conor Simpson]

Hi Group

Firstly, I would like to take this opportunity to thank Prof. Broman for r/QTL and all the accompanying resources you so freely provide.

Secondly, apologies for the long-winded question you are about to read.

I am constructing a map using markers with known positions. To investigate what markers might be switched I generated new linkage groups using "formLinkageGroups()". This resulted in some chromosomes being split into two or three linkage groups.

For example. Chromosome 5 was split into three linkage groups, LG 9, 22 and 23, each of which had the following order (marker names are based on the order they occur on the physical chromosome):

Linkage group 22:

Sc5_16, Sc5_17, Sc5_18, Sc5_19, Sc5_20, Sc5_23, Sc5_24, Sc5_26, Sc5_30, Sc5_31, Sc5_32, Sc5_33, Sc5_34, Sc5_35, Sc5_36, Sc5_37, Sc5_39, Sc5_40, Sc5_41, Sc5_42, Sc5_43, Sc5_44, Sc5_46, Sc5_53, Sc5_133, Sc5_134, Sc5_135, Sc5_136, Sc5_137, Sc5_138, Sc5_139

Linkage group 9:

Sc5_52, Sc5_54, Sc5_55, Sc5_56, Sc5_57, Sc5_59, Sc5_63, Sc5_64, Sc5_67, Sc5_68, Sc5_69, Sc5_70, Sc5_71, Sc5_72, Sc5_73, Sc5_74, Sc5_75, Sc5_80, Sc5_82, Sc5_83, Sc5_84, Sc5_86, Sc5_87, Sc5_88, Sc5_91, Sc5_92, Sc5_93, Sc5_98, Sc5_100, Sc5_101, Sc5_102, Sc5_103, Sc5_105, Sc5_106, Sc5_107, Sc5_109, Sc5_110, Sc5_111, Sc5_112, Sc5_116, Sc5_117, Sc5_120, Sc5_122, Sc5_123, Sc5_124, Sc5_126, Sc5_127, Sc5_128, Sc5_129, Sc5_130, Sc5_131, Sc5_149, Sc5_150, Sc5_151, Sc5_157, Sc5_158, Sc5_159, Sc5_160, Sc5_161, Sc5_162, Sc5_164, Sc5_165, Sc5_166, Sc5_167, Sc5_168, Sc5_170, Sc5_171, Sc5_172, Sc5_178, Sc5_182, Sc5_183, Sc5_184, Sc5_185, Sc5_187, Sc5_192, Sc5_193, Sc5_194, Sc5_195, Sc5_196, Sc5_197, Sc5_201, Sc5_202, Sc5_203, Sc5_204, Sc5_206, Sc5_218, Sc5_223, Sc5_229, Sc5_231, Sc5_232, Sc5_234, Sc5_239, Sc5_240, Sc5_244, Sc5_245, Sc5_251, Sc5_252, Sc5_253, Sc5_254, Sc5_256, Sc5_270, Sc5_276, Sc5_280, Sc5_281, Sc5_283, Sc5_284, Sc5_285, Sc5_286, Sc5_287, Sc5_288, Sc5_289, Sc5_291, Sc5_292, Sc5_293, Sc5_294, Sc5_295, Sc5_296, Sc5_297, Sc5_298, Sc5_299, Sc5_301, Sc5_302, Sc5_303, Sc5_304, Sc5_307, Sc5_308, Sc5_309, Sc5_310, Sc5_311, Sc5_312, Sc5_313, Sc5_314, Sc5_317, Sc5_318, Sc5_319, Sc5_320, Sc5_321, Sc5_322,
Sc5_330, Sc5_331, Sc5_333, Sc5_334, Sc5_336, Sc5_337, Sc5_338, Sc5_339, Sc5_343, Sc5_347, Sc5_350, Sc5_351, Sc5_352, Sc5_353, Sc5_354, Sc5_355, Sc5_356, Sc5_357, Sc5_360, Sc5_361, Sc5_363, Sc5_364, Sc5_365, Sc5_367, Sc5_369, Sc5_370, Sc5_371, Sc5_372, Sc5_373, Sc5_376, Sc5_377, Sc5_378, Sc5_381, Sc5_386, Sc5_389, Sc5_393, Sc5_394, Sc5_395, Sc5_397, Sc5_398, Sc5_399, Sc5_402, Sc5_403, Sc5_404, Sc5_406, Sc5_407, Sc5_408, Sc5_409, Sc5_410, Sc5_411

Linkage group 23:

Sc5_414, Sc5_415, Sc5_416, Sc5_417, Sc5_418, Sc5_419, Sc5_420, Sc5_421, Sc5_422, Sc5_423, Sc5_424, Sc5_425, Sc5_426, Sc5_427, Sc5_428, Sc5_429, Sc5_430, Sc5_431, Sc5_432, Sc5_434, Sc5_435, Sc5_439, Sc5_440, Sc5_442, Sc5_443, Sc5_447, Sc5_449, Sc5_450, Sc5_451, Sc5_452

I have attached the recombination frequency/LOD heat-map of these linkage groups and an individual plot of recombination frequency/LOD for an individual marker that is representative of what I see for any marker in linkage group 22 and 23.

It is clear from above that the groups are formed of markers that are at either end of the chromosome (LG-22 and LG-23) and the core of the chromosome (LG-9). I have also colour coded the location in the core linkage group (LG-9) where these other markers should belong.

When I look at the two-locus genotype tables (using " geno.crosstab()"). For markers in each group they obviously satisfy each other but appear "switched" when compared to LG-9.

My question is what to do here. Because the heat-map indicates to switch these markers. But is it not likely that these are potentially indicating recombination hot-spots or that the chromosome is long enough to be considered as three separate chromosomes? I have encountered this sort of thing before and often I just remove the alleles that appear to be switched. But here this is quite a few markers and would mean eliminating whole regions of the chromosome in question from my map.

Even focusing on the markers I highlighted in red. These may appear great candidates to switch but they do not look so when compared within themselves. I think maybe I have gotten confused somewhere but I do not want to make a blunder in my map making here so have come to the group for help.

Any comments, thoughts or suggestions would be greatly appreciated.

Thanks,

Conor

[Conor Simpson]

I just realised I should state that I have not actually ordered the markers for this chromosome using est.map() or ripple() hence the order here looks so neat (at least when printed out if not in the heat-map)

[Karl Broman]

I'm not sure I understand what you're trying to do, but it seems like the linkage map construction part is not worth the effort, and that it might be better to find a more direct way to solve the problem.

You say you are "constructing a map using markers with known positions. To investigate what markers might be switched..."

I guess I take it that you have a problem with some markers being out of phase with the surrounding ones?

The chromosome assignments and marker order are really useful to identify problem markers, so I'd leave them where they are and think of other ways to identify which markers need correction and how.

karl

[Conor Simpson]

Hi Karl,

Yes, my problem is that groups of markers appear to be out of phase with others.

What is the best thing to do with markers that are so out of phase? Because surely they are not errors and should not be removed or switched but they will also greatly increase the length of the chromosomes if left in.

Best,

Conor

[Karl Broman]

You need to somehow figure out which are the problem markers and fix those.

But it seems like the best information for that would come with the markers in their physical order and positions.

karl

[Conor Simpson]

OK I will continue to work on it - thank you,

Conor

[Conor Simpson]

Hi Karl,

I am getting incredibly confused on the topic of allele switching. I have worked through your guide and have searched through this group with the most useful explanation coming from you in the following discussion:

https://groups.google.com/g/rqtl-disc/c/jpDZraTrApc

However, I am still confused. As I have so many apparently switched alleles (see attached heat map). I am unsure how best to deal with them. How do I know which ones are correct? I have the parental genotypes available but I do not see how these can help me as they match up to what I see in the heatmaps.

My interpretation of my example here is that chromosome 4 has quite a few switched markers but these are still largely outnumbered by the correct ones and I should switch all of these and move on. However chromosome 7 looks completely riddled with switched markers and these are in relatively the same proportion as the non-switched markers so I should eliminate chromosome 7 to be safe, which is obviously not what I want to do.

Any thoughts and explanations you have on this would be appreciated.

Thanks,

Conor

[Conor Simpson]

Attachment

[Karl Broman]

I don't understand it either.

I understand that you are working with an intercross from two inbred founders. Genotypes are encoded as AA, AB, BB where A is the allele from one founder and B is the allele from the other founder.

The alleles being "switched" indicates that the genotypes of the founders were incorrect...that the A and B alleles are coming from the opposite parents and that the genotypes need to be re-encoded with AA -> BB and BB -> AA.

I would expect just a small proportion of markers (1-5%)  to have this problem, and not half.

karl

[Conor Simpson]

Thanks for a swift response - you understand correctly, and I am oddly reassured that this is not a common problem and therefore likely an issue with the genotype data. Have you ever come across data as dodgy as this? If so what did you do?

I was thinking of potentially removing the markers for chromosome 4 and then making two maps - one that includes the markers that appear "correct" for chromosome 7 and one that considers only the ones that are seen as switched in this instance. I would then perform my mapping and simply note that chromosome 7 can't be trusted.

What are your thoughts?

Best,

Conor

[Karl Broman]

I don't really want to speculate, but from the pictures you've shown, I wouldn't conclude that the data are dodgy or have a major problem; it seems instead like some misunderstanding that can be fixed.

Many people think of QTL mapping from the lens of GWAS and don't think about encoding genotypes according to founder origin, but rather like A = major allele and B = minor allele.

I would go back to the source, and also ask further about the genotypes of the founder lines.

karl

[Conor Simpson]

Hi Karl,

I went back and carefully checked the raw genotyping data, it is definitely encoded correctly, with A being the allele from parent A and B being from parent B. There did turn out to be quite a few markers that should have been removed - i.e. markers that did not differ between parents or were not heterozygous in the F1s.

I removed all such markers and re-constructed the map as before. This resulted in a large loss of total markers for most chromosomes. However, the problem chromosomes (2, 4 and 7) still look pretty much as they did before. I do not now what is going on - I am thinking there maybe an underlying issue with the genome assembly from which these markers were positioned and it may be best to try this again on a previous assembly.

Best,

Conor