Activity for Pasi Rastas
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Pasi Rastas posted a comment on discussion General Discussion
To add, in Filtering2, heterozygoteRate should be set to some small value (like 0.01). Cheers, Pasi
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Dear Xochitl Granados, Thank you for your question. I think this would be easiest to analyse as F1. Changing population (diploid) genotypes as 0/0 => 0/0 and 1/1=>0/1 and keeping mother as such. However, this requires quite much scripting to re-format the data, depending on which format your data is (for example, population variants could be called haploid). Maybe ParentCall2 could support this in the future. Without reformatting the (diploid called) population data, it can be analysed with two dummy...
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Thanks Hongbo, for pointing the missing file. The file is now added. Cheers, Pasi
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Please read the part about selfing crosses on the wiki as well. Cheers, Pasi
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java locale fix
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Dear both, I think you can analyse such data as F2 by setting P1-P4 to grandparents, adding dummy parents and then F7 as offspring. Cheers, Pasi
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Dear Nick, There should be documentation in the wiki now about linkage mapping without parents. Please ask if it is not clear. However, it might be possible that your data can be analysed without any of these tricks. Just analyse the data as known parents, the sex averaged map will work anyway... Cheers, Pasi
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There should be documentation in the wiki now. Please ask if it is not clear. Cheers, Pasi
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Dear Farid, Sorry, I don't know any software to do this. For best results, I would run SeparateChromosomes2 on all markers, and only then would do the thinning of markers in the linkage groups so that the markers selected will be contributing to the map. Probably good result would be obtained by thinning markers after ParentCall2 or Filtering2 on all markers. Cheers, Pasi
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LM3 Home
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Dear Farid, For the first question, I think it due to fact that Filtering2 will remove some distorted markers (dataTolerance=0.001) and after this, some of them might become non-informative. So ParentCall2 will do this filtering without taking into account the distortion. Typically I don't include removeNonInformative in Filtering2 as then I can easily use datasets with different dataTolerance values, if needed, as the number of markers stays the same. The second one: Yes, you can use your genome...
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Dear Nick, Thank you for your question and sorry for taking some time to answer as I was on holiday. I think you can construct the map as if there were no parental data. This has been in the discussion earlier. I will add the information to the wiki soon. If you are in a hurry, please check the wiki and the old discussion threads. Cheers, Pasi
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LM3 Home
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Dear Jess, This is very odd indeed. Note that the map position of a marker can be impossible to determine exactly. You can output the map position intervals from OrderMarkers2 (calculateIntervals=file) to see the uncertainty in the map positions. You can also try parameter usePhysical (you can tinker it to find a suitable value) to have a map between the physical order and "de novo". Cheers, Pasi
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Dear Jess, Nice to hear from your progress! The red lines in the first LMPlot are indicating a loop in the graph. It means that there is a recurring crossover in this region. It could be an inversion between the reference order and the map order. As there is only one recurring crossover, this is not a big deal, it only adds two extra crossovers (at most, as this could be correct solution as well). The second one is harder to figure out (if you run LMPlot again, it will untangle the graph in a different...
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Dear Jess, Nice to hear from your progress! The red lines in the first LMPlot are indicating a loop in the graph. It means that there is a recurring crossover in this region. It could be an inversion between the reference order and the map order. As there is only one recurring crossover, this is not a big deal, it only adds two extra crossovers (at most, as this could be correct solution as well). The second one is harder to figure out (if you run LMPlot again, it will untangle the graph in a different...
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Dear Jesi, You can merge and split linkage groups in the process as you like: #merge lgs 1 & 2 awk '(($4==1 || $4==2) && $2=="CHR1")' numeric_snsp.txt >order1.phys #split lg 3 awk '($4==3 && $2=="CHR3")' numeric_snsp.txt >order3.phys awk '($4==3 && $2=="CHR4")' numeric_snsp.txt >order4.phys Maybe it is best to figure out how the LGs and chrs correspond to each other and why they are not corresponding 1 to 1. Cheers, Pasi
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Dear Jesi, The example is indeed for creating both, the de novo map and the physical map. The order1.input is the denovo map for Lep-Anchor. However, you need the linkage groups only for the physical order (you can try to put all markers in a chromosomal assembly but this might cause problems as there are very likely some "jumping" markers). If you have the snp names (snps.txt) and linkage groups from SeparateChromosomes2 (map_lg.txt) you can try something like this: paste snps.txt map_lg.txt|awk...
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Dear Jesi, The example is indeed for creating both, the de novo map and the physical map. The order1.input is the denovo map for Lep-Anchor. However, you need the linkage groups only for the physical order (you can try to put all markers in a chromosomal assembly but this might cause problems as there are very likely some "jumping" markers). If you have the snp names (snps.txt) and linkage groups from SeparateChromosomes2 (map_lg.txt) you can try something like this: paste snps.txt map_lg.txt|awk...
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Dear Jesi, The example is indeed for creating both, the de novo map and the physical map. The order1.input is the denovo map for Lep-Anchor. However, you need the linkage groups only for the physical order (you can try to put all markers in a chromosomal assembly but this might cause problems as there are very likely some "jumping" markers). If you have the snp names (snps.txt) and linkage groups from SeparateChromosomes2 (map_lg.txt) you can try something like this: paste snps.txt map_lg.txt|awk...
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Dear Jesi, The example is indeed for creating both, the de novo map and the physical map. The order1.input is the denovo map for Lep-Anchor. However, you need the linkage groups only for the physical order (you can try to put all markers in a chromosomal assembly but this might cause problems as there are very likely some "jumping" markers). If you have the snp names (snps.txt) and linkage groups from SeparateChromosomes2 (map_lg.txt) you can try something like this: paste snps.txt map_lg.txt|awk...
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Dear Farid, Thank you for your question. The parameter combo evaluateOrder and improveOrder=0 is the way. The order must be given with the numerical identifiers. Lep-Anchor wiki contains some examples of this: http://sourceforge.net/projects/lep-anchor . Cheers, Pasi
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Dear Dave, Thank you for your question. Typically you end up with many small groups and hopefully desired number of large groups. If you end up collapsing two or more groups, you can re-run SeparateChromosomes2 again with the map parameter giving the previous result (and a higher lodLimit). The collapsed group is most likely the largest (lg=1), but if it is not the parameter lg can be given. The small groups can be removed manually or with the parameter sizeLimit. Cheers, Pasi
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Dear Dave, Thank you for your question. Typically you end up with many small groups and hopefully desired number of large groups. If you end up collapsing two or more groups, you can re-run SeparateChromosomes2 again with the map parameter giving the previous result (and a lower lodLimit). The collapsed group is most likely the largest (lg=1), but if it is not the parameter lg can be given. The small groups can be removed manually or with the parameter sizeLimit. Cheers, Pasi
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wrappers with markerSupport, ChainPaf
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Dear Dave, Thank you for your question. Typically you end up with many small groups and hopefully desired number of large groups. If you end up collapsing two or more groups, you can re-run SeparateChromosomes2 again with the map parameter giving the previous result. The collapsed groups is most likely the largest (lg=1), but if it is not the parameter lg can be given. The small groups can be removed manually or with the parameter sizeLimit. Cheers, Pasi
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Dear Lorenzo, I think your version of LM3 is recent enough. The problem you are having is probably due to not using the same phasing for LOD computation and map evaluation. Safer option is to calculate LOD scores for each 10 runs and pick the LOD result based on the likelihoods (instead of evaluateOrder). You could also use the map from evaluateOrder output. The parameter phasingIterations=3 might improve the evaluated order's phasing... Cheers, Pasi
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Dear Lorenzo, Thank you for your question. I did not find any obvious problem with the code for calculating unique map positions (this routine is only used in the LOD calculation). I even tried in on real data, I got 147 unique positions from the LOD and from the map. Are you using the latest version of LM3? Some older versions used identicalLimit parameter in finding these unique positions. The LOD output contains the marker names, did you check the positions for these markers from the map? Cheers,...
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Dear Lorenzo, Thank you for your question. I did not find any obvious problem with the code for calculating unique map positions (this routine is only used in the LOD calculation). I even tried in on real data, I got 147 unique positions is LOD and from the map. Are you using the latest version of LM3? Some older versions used identicalLimit parameter in finding these unique positions. The LOD output contains the marker names, did you check the positions for these markers from the map? Cheers, P...
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Dear Farid, Thanks for noting this. I will try to test the software in windows as well. This might also help: you can (maybe) add -Dline.separator=$'\n' to java command to set the end of line separator to \n. Or to -Dline.separator=$'\r\n' if it then works. Cheers, Pasi
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Dear Yess Alvarez, Normally you just construct one map, it will have male and female position for each marker. You can construct separate male and female maps as well, but you should first try with all markers. Cheers, Pasi
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Dear Farid, Thank you for your question and sorry to hear you about your problem with Lep-MAP3. I cannot find anything wrong in your commands, they seem ok. The error message is originating from the part where the data is read and the marker name is extracted. Lep-MAP3 does not find two tab separated fields from the line. I cannot say exactly what the problem is. Maybe it is the end of line characters (\r\n) in dos? I know that some windows text editors can fix/change these (I haven't used windows...
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Dear Farid, Thank you for your question and sorry to hear you about your problem with Lep-MAP3. I cannot find anything wrong in your commands, they seem ok. The error message is originating from the part where the data is read and the marker name is extracted. Lep-MAP3 does not find two tab separated fields from the line. I cannot say exactly what the problem is. Maybe it is the end of line characters (\r\n) in dos? I know that some windows text editors can fix/change these. (I haven't used windows...
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Dear Tianzhu, The parameter minError controls how much you trust your data. It will cap the genotype quality to this value, 0.1 is phred score of 10. If this is RADseq data, very useful parameter is proximityScale, value of 50 or 100 might work for most data to scale down the effect of too close by SNPs (from the same RAD site). I would be more interest on how you generated the input genotype data. Maybe the problem is there? Cheers, Pasi
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Dear Yutang, Yes, it is consistent. Cheers, Pasi
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Dear Yutang, In the phase information, first there is the paternal phased genotype (e.g. GA) and then, separated by "," is the maternal one (each family separated by ";"). The two haplotypes are in arbitrary order (but you know that they are the fathers two haplotypes and the mothers two haplotypes). If you use grandparents for phasing, then the first is inherited from the parent's father and second from the parent's mother. Cheers, Pasi
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Dear Yutang, In the phase information, first there is the paternal phased genotype (e.g. GA) and then, separated by "," is the maternal one (each family separated by ";"). The two haplotypes are in arbitrary order (but you know that they are the fathers two haplotypes and the mothers two haplotypes). If you use grandparents for phasing, then the first is the parent's father's and second the parent's mother's. Cheers, Pasi
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Dear Yutang, In the phase information, first there is the paternal genotype (e.g. GA) and then, separated by "," is the maternal one (each family separated by ";"). The two haplotypes are in arbitrary order (but you know that they are the fathers two haplotypes and the mothers two haplotypes). If you use grandparents for phasing, then the first is the parent's father's and second the parent's mother's. Cheers, Pasi
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Dear Yutang, Nice to hear from you. And thanks for the suggestion, the phase information has been very useful. I added it soon after the PAG meeting where we discussed it. I have used the phase for so many projects, that I would not want to change the format anymore. There is the vcfPhase.awk script (in scripts.zip) that adds "|" between alleles for vcf input (AT=>0|1). This could be modified to change AT=>A|T. Note that the ACGT codes are correct only for the Lep-MAP3 variant calling. For vcf input,...
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Dear Tianzhu, I think this is about the quality of your data and number of markers. I would aim for a higher number of markers, often you get about 1000 markers per chr with RADseq and 200 markers is still quite few. Moreover, I have no idea how you have analysed and processed your data, these are critical steps in the map construction. Cheers, Pasi
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Dear Tianzhu, Aha. We are talking about different thing here. I thought there was 0.7 fraction of crossovers between two adjacent markers (rf in the mapping functions). The map is about 75 cM. It is within my expected range of 50-80 cM. If you think this is too long, check if there are individuals that recombine too many times over all chrs. I think you can get this information from the Lep-MAP3's error stream output. You can also force Lep-MAP3 to produce shorter maps by changing the scale parameter....
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Dear Tianzhu, Aha. We are talking about different thing here. I thought there was 0.7 fraction of crossovers between two adjacent markers (rf in the mapping functions). This makes sense. The map is about 75 cM. It is within my expected range of 50-80 cM. If you think this is too long, check if there are individuals that recombine too many times over all chrs. I think you can get this information from the Lep-MAP3's error stream output. You can also force Lep-MAP3 to produce shorter maps by changing...
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Dear Tianzhu, Could you post the map ends from Lep-MAP3, and maybe the commands you have used? I have not seen over 0.5 recombination fractions. Are you using grandparents for phasing? Without grandparents, I think the Lep-MAP3 phasing algorithm should flip 0.7 in this case to 0.3. Proximityscale should be about the length of the RAD site. Value of 100 (in bp) should be ok for most cases. Cheers, Pasi
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Dear TZ, Thank you for your question. My maps for Lepidoptera has been 50-80cM. If there are poor markers, these tend to go to the map ends. Maybe this is the case here. Removing markers from the ends might solve the problem. The new default mapping function is Morgan, so recombination rate of 0.7 produces 70cM gaps. For other mapping functions, this would break the linkage group. Also check that the data correct, for example IBD values between parents and offspring are about 0.5 or higher. If your...
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Dear chinj cheung, Lep-MAP3 requires data with both parental genotypes. ParentCall2 tries to impute missing parents if it is possible. This imputation is difficult if you have only few offspring and no grandparents or half-sib families. Family limit defines how certain the imputed parental genotypes must be. Lowering it might yield more data but there might be more errors as well. I have constructed good maps with ignoreParentOrder as well. This might help more if both parents are missing as it keeps...
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Dear chinj cheung, Wow, this is a huge dataset! If you have a lot of small families with only one parent, it might happen that the parental genotypes cannot be inferred. In this case you also obtain all 1s. You can try to lower familyLimit parameter (try 1 or 1.5) if this is the case. For a single parent family, you might need 5-8 offspring to call the missing parental genotype. If you have halfsib families, they might help. Also the parameter ignoreParentOrder in ParentCall2 can be tried but that...
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Dear chinj cheung, Are you sure that the individual names match in your pedigree and vcf? If not, ParentCall2 will give a warning that the individual(s) are not found and are set to all missing... This would explain all 1s (missing) in your data. ParentCall2 should read GT field if it does not find PL or GL. Cheers, Pasi
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Dear Julien, JoinSingles2All lists also alternative linkage groups with their LOD scores. So marker is put into LG 34 with LOD=23.8 but it could be in 3 or 4 (with much lower LOD, < 5). This information is not used by other modules but sometimes it is good to check. It can be useful for example when analysing very small datasets (say 8-20 individuals). Cheers, Pasi
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Dear chinj cheung, Yes, you should include halfSibs=1 to ParentCall2 if you have shared parents. You can add the same grandparent in multiple families, just add the different family ID for each column with the same grandparent. Lep-MAP3 does not use the information of same grandparents, it is handled the same way whether the grandparents are unique to a family or not. Cheers, Pasi
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Dear chinj cheung, Thank you for your question. You should include both families in one pedigree. The attached pedigree works for me, and ParentCall2 gives the hint "parent P1_m1 occurs 2 times in the pedigree use halfSibs=1 to utilize this information". Cheers, Pasi
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The file is corrupted (due to nohup?). The ParentCall2 output from out and error streams are mixed. Run ParentCall2 without nohup (or add extra parenthesis so that this does not occur). Cheers, Pasi
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Hi Julien, Can you share a few lines of your parentTest.txt ? Like 10-20 first lines? Cheers, Pasi
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Hi Julien, Can you share a few lines of your parentTest.txt ? Cheers, Pasi
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Dear Julien, The Parenttest.txt might be a wrong file for this purpose. How did you produced it? Based on the name, I would think it is not the ouput from ParentCall2? Cheers, Pasi
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Dear Julien, Could you send me the Filtering2 command line you have run? Cheers, Pasi
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Dear Xochitl Granados, Sorry to hear about your problems. First of all, the map could be totally ok. Without the physical coordinates it is difficult say for sure. For the first group, LMPlot does not show any obvious problems. The map length for this is quite long but it could be real. Secondly, I noticed that you have all of your linkage groups in the same file (order1scale.txt). It is probably easier to separate linkage groups. You can split the file after LG = 1 ... LG = 12. I find it easier...
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FitStepFunction
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I am happy to help, Cheers, Pasi
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Dear Julien, Thank you for your question, you are not annoying at all! It seems the individual names in the vcf and pedigree do not match. All the names in the vcf have underscore _ like 40821467912064082140401641_4112182101227 but the names in pedigree don't have it. As such, no data from the vcf file is obtained. To fix this problem, you have to figure the correct individual names. Cheers, Pasi
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Dear Julien, Thank you for your question, you are not annoying at all! It seems the individual names in the vcf and pedigree do not match. All the names in the vcf have underscore _ like 40821467912064082140401641_4112182101227 but the names in pedigree don't have it. As such, no data from vcf file is obtained. To fix this problem, you have to figure the correct individual names. Cheers, Pasi
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Dear Marina, As Lep-MAP3 imputes data on all markers, P2 is always "1 2" after map2genotypes.awk but the genotype of P2 is not necessary always heterozygous. If you want to see the parental genotypes, check the phase information from the output map. "--" indicates that the parent is homozygote. If you use the map2genotypes_phased.awk script, then imputed genotype has alleles 1 or 2, but ones inherited from the parents are coded with A, C, G or T. Cheers, Pasi
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Dear Marina, Thank you for your question. Sorry, but I don' fully understand the problem here. Lep-MAP3 should be able to call markers where one or both parents are heterozygous. Do you have data on all individuals GP1...4 and P1 and P2? If the data is uncertain about which of the parents is heterozygous (when exactly one parent is), it does not pass ParentCall2 without ignoreParentOrder flag. Typically this flag is used on datasets not containing any parental genotypes (and has to be handled in...
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Dear Marina, Thank you for your question. I don't think you have to change the recombination parameters. If the data quality would be poor you might want to change them. You are happy to test changing these parameters (e.g. recombination1=0.0007, recombination2=0.0013), I am only doubting they make any difference. Cheers, Pasi
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Dear Joey, The ParentCallPloidy has been uploaded now. You have to provide parameter ploidy=4 to Pileup2Likelihoods to call tetraploid markers. The ParentCallPloidy should work as ParentCall2, note the parameter ploidy=4 (default) as well. Finally, you can convert the data to diploid using convert2diploid.awk script (should be in scripts.zip), this also has parameter ploidy (default=4, and check that parents are the last individuals, and if not change parameters father and mother accordingly or change...
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Dear Joey, The ParentCallPloidy has been updated now. You have to provide parameter ploidy=4 to Pileup2Likelihoods to call tetraploid markers. The ParentCallPloidy should work as ParentCall2, note the parameter ploidy=4 (default) as well. Finally, you can convert the data to diploid using convert2diploid.awk script (should be in scripts.zip), this also has parameter ploidy (default=4, and check that parents are the last individuals, and if not change parameters father and mother accordingly or change...
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ParentCallPloidy
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Dear Joey, The vcf file has tetraploid calls. ParentCall2 does not support them. Sorry. Earlier I have made ParentCallPloidy to work with polyploid (simplex) markers (see https://pubmed.ncbi.nlm.nih.gov/32004453/). However, it does not support vcf files, only Pileup2Likelihoods output. I noticed that ParentCallPloidy is not in available. I can add it Lep-MAP3 if you want to use it? Another option would be to convert the simplex markers to diploid (in the vcf) and use that for Lep-MAP3. Lep-MAP3 only...
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Dear Julien, Yes, you have to have have each full sib family separately with a unique family id. ParentCall2 will detect and utilise half-sib families from common parents. Other modules do not benefit from half-sib information. Cheers, Pasi
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Dear Julien, I think you have to provide the vcf file like this: java ParentCall2 data=pedigree.txt vcfFile=test.vcf Where the pedigree file is given via data parameter. Cheers, Pasi
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Dear Xochitl Granados, That does not look good... How do you map the marker numbers to the physical coordinates? Lep-MAP3 renames markers as 1...N, in your case 1...1280821. These should be mapped back to the physical coordinates: Typically I find first the SNP names as something like this (modified from the Lep-Anchor wiki): awk '(NR>=7){print $1"\t"$2}' dataAll_fil.call >snps.txt awk '(NR==FNR){map[NR-1]=$0}(NR!=FNR && /^[^#]/){print map[$1],$2,$3}' snps.txt order_wp1.txt >order_wp_1.mapped And...
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Dear Ross, I found the problem! It is the empty line in your pedigree file. Try this file. Cheers, Pasi
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Dear Ross, Could you post the first line(s) of the post.out as well? Cheers, Pasi
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Dear Ross, The Pileup2Likelihoods works with samtools mpileup output (input consists of individual bam files). ParentCall2 can input vcf directly via vcfFile parameter. Cheers, Pasi
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Dear Dustin, Thank you for interesting question. There is no way to separate male and female maps for selfing cross from genotype data. The crossovers are put more or less arbitrary by OrderMarkers2 as it has independent male and female models. Just average the map positions and you should be fine. Cheers, Pasi Ps. Please remember to use selfingPhase=1 or grandparentPhase=1 parameters with selfing data.
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Dear Dustin, Thank you for interesting question. There is no way to separate male and female maps for selfing cross from genotype data. The crossovers are put more or less arbitrary by OrderMarkers2 as it has independent male and female models. Just average the map positions and you should be fine. (Due to asymmetry in the haplotype model in LM3, haplotypes without crossovers are preferred so the male map could be shorter by having more 0 crossovers) Cheers, Pasi Ps. Please remember to use selfingPhase=1...
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small fixes
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Dear Haley, It seems you have the parents of family 1 also as offspring in the same family: 1 C1_5_3_2_3_BM 0 0 1 0 1 C1_5_3_2_3_BM C1_5_3_2_3_BM C1_5_3_2_3_BF 1 0 1 C1_5_3_2_3_BF 0 0 2 0 1 C1_5_3_2_3_BF C1_5_3_2_3_BM C1_5_3_2_3_BF 2 0 (in transpose) So the same parental individuals are used as grandparents as well... I think this does not matter but it is clearly not correct. So please remove these extra lines and you are fine. Cheers, Pasi
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Dear Haley, It seems you have the parents of family 1 also as offspring in the same family: 1 C1_5_3_2_3_BM 0 0 1 0 1 C1_5_3_2_3_BM C1_5_3_2_3_BM C1_5_3_2_3_BF 1 0 1 C1_5_3_2_3_BF 0 0 2 0 1 C1_5_3_2_3_BF C1_5_3_2_3_BM C1_5_3_2_3_BF 2 0 (in transpose) So the same parental individuals are used as grandparents as well... I think this does not matter but it is not clearly correct. So please remove these extra lines and you are fine. Cheers, Pasi
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Dear Haley, Thank you for your question. The part of the pedigree showing does not contain grandparents. Could you please post your entire pedigree so I could check it easily? Cheers, Pasi
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Dear Haley, Thank you for your question. The part of the pedigree showing does not contain grandparents. Could you post your entire pedigree so I could check it easily. Cheers, Pasi
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Dear Marina, Please check that the grandparents are related to the parents (e.g. IBD module, or some other sofware), and also relatedness of parents and offspring. This is critical for linkage mapping to work! If the phasing does not work even if you are sure of the grandparents, please let me know. Cheers, Pasi
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Dear Marina, Thank you for your question. A linkage group is split into several pieces (X, X.1, X.2, ...) if the mapping function gives infinity distance (recombination fraction of >= 50%). Here it looks like the phasing is wrong, the markers are in the opposite phase between blocks LG=0 and LG=0.1. And you are using the grandparents for phasing (grandparentPhase=1). Maybe the grandparents are incorrect? Could you please give a little more information what you have done (and would like to do) here?...
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Dear Etienne, Great that I could be of help. And sorry, Lep-MAP3 does not have any other documentation than this forum and the program itself. I feel that keeping such a documentation up to date would be too time consuming... And it is very nice that users are not afraid to ask if they are unsure:) Cheers, Pasi
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Dear Tami, Thank you for your question. You don't need grandparents, Lep-MAP3 phases according to the parents then. It does not affect analysis for a single family cross. Cheers, Pasi
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Dear Etienne, Thank you for you question. I think it is normal that order likelihood is lower if you evaluate the map in the physical order compared to denovo order. If the physical order is correct, using it is easy way to get good maps. Things to check with physical maps: 1) check for outlier individuals based on the number of crossovers (recobinations). Lep-MAP3 ouputs these in the error stream. 2) increase phasingIterations, e.g. to 3. If you notice a change in the likelihood the phasing has...
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Dear Etienne, Thank you for you question. I think it is normal that order likelihood is lower if you evaluate the map in the physical order compared to denovo order. If the physical order is correct, using it is easy way to get good maps. Things to check with physical maps: 1) check for outlier individuals based on the number of crossovers (recobinations). Lep-MAP3 ouputs these in the error stream. 2) increase phasingIterations, e.g. to 3. If you notice a change in the likelihood the phasing has...
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ChainPaf fix and speedup
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