# Problem with split.abund and group file **URL:** \ **Category:** Commands in mothur\ **Created:** [September 3, 2014, 2:03pm UTC](https://forum.mothur.org/t/problem-with-split-abund-and-group-file/2003 "2014-09-03T14:03:24Z")\ **Posts on this page:** 2\ **Page:** 1
**Author:** ![svazquez](https://avatars.discourse-cdn.com/v4/letter/s/f6c823/32.png) [@svazquez](https://forum.mothur.org/u/svazquez)\ **Post date:** [September 3, 2014, 2:03pm UTC](https://forum.mothur.org/t/problem-with-split-abund-and-group-file/2003/1 "2014-09-03T14:03:24Z")
Hi I am working 454 data and have to remove the OTUs with only 1, 2 or 3 seqs (yes, I know is not the best choice, but I was asked to do like this, I’m sorry). But also I have to get the ordination (PCoA) based on unifrac distances. Of course this last and the other results (which will be based on the OTU approach) have to be based on the same dataset (same fasta file, type and number of sequences per sample). I followed the 454 SOP up to the cluster step and got the final4.an.list, together with my final4.fasta, final4.groups and final4.names files. Then, split.abund(fasta=final4.fasta, list=final4.an.list, group=final4.groups, cutoff=3, label=0.03) Output File Names: final4.an.0.03.rare.list final4.an.0.03.abund.list final4.0.03.rare.groups final4.0.03.abund.groups final4.0.03.rare.fasta final4.0.03.abund.fasta summary.seqs(fasta=final4.0.03.abund.fasta, name=final4.names) Start End NBases Ambigs Polymer NumSeqs Minimum: 1 1571 493 0 3 1 2.5%-tile: 1 1573 500 0 4 4608 25%-tile: 1 1573 505 0 4 46073 Median: 1 1573 520 0 5 92145 75%-tile: 1 1573 527 0 5 138217 97.5%-tile: 1 1573 529 0 7 179681 Maximum: 3 1573 574 0 8 184288 Mean: 1.00001 1573 517.172 0 4.94459 # of unique seqs: 38482 total # of seqs: 184288 make.shared(list=final4.an.0.03.abund.list, group=final4.0.03.abund.groups, label=0.03) count.groups() 10A\_11 contains 7563 5A\_10 contains 6590 11A\_10 contains 7117 5A\_11 contains 6302 11B\_10 contains 6154 6A\_11 contains 7735 11C\_10 contains 6696 6B\_11 contains 7408 12A\_10 contains 5950 7A\_10 contains 5736 12B\_10 contains 8900 7A\_11 contains 8435 1A\_11 contains 6508 7B\_10 contains 7686 1B\_11 contains 7614 8A\_10 contains 6151 2A\_11 contains 8065 8A\_11 contains 6781 2B\_11 contains 7924 8B\_10 contains 5481 3B\_11 contains 6144 9A\_10 contains 6463 4A\_11 contains 5278 9A\_11 contains 6509 4B\_11 contains 6270 S1B1S\_10 contains 5767 Total seqs: 184288 S1B1S\_11 contains 7061 sub.sample(shared=final4.an.0.03.abund.shared, size=5278) classify.otu(list=final4.an.0.03.abund.list, name=final4.names, taxonomy=final4.taxonomy, label=0.03) Up to here, all seem to have worked well!! I could get the rarefaction curves, diversity indexes, heatmap and so on from the subsampled.shared file. Then, when I tried to do the PCoA based on unifrac distances: dist.seqs(fasta=final4.0.03.abund.fasta, output=phylip, processors=10) clearcut(phylip=final4.0.03.abund.phylip.dist) I tried: unifrac.unweighted(tree=final4.0.03.abund.phylip.tre, name=final4.names, group=final4.0.03.abund.groups, distance=lt, processors=10, random=F, subsample=T) It didn’t work and a lot of seqs names were listed saying that those seqs were not in my groups file. I tried with the first group file (before split.abund): unifrac.unweighted(tree=final4.0.03.abund.phylip.tre, name=final4.names, group=final4.groups, distance=lt, processors=10, random=F, subsample=T) Setting subsample size to 5830 Now it worked but the size of subsample was higher than that I had to set for subsampling the shared file. :roll: Why is not working if the fasta file is the same and the groups file should match it, as both were outputs after split.abund?? Sorry, but I cannot see where I am doing sth wrong :oops: Thanks!! ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [September 10, 2014, 5:51pm UTC](https://forum.mothur.org/t/problem-with-split-abund-and-group-file/2003/2 "2014-09-10T17:51:36Z")
You might want to include the names file in the split.abund command.