Inconsistant subsampling

[campenr]

Hello,

First I'm raising this as an issue separatley from #311 as although they both are about mothurs subsampling the root cause may be unrelated.

I am finding that I get inconsistant results with summary.single if I pre subsample to a set size (in my case 12,000 sequences) using sub.sample(), or if I let summary.single do the sub sampling with subsample=12000.

Some of my samples are being subsampled from more than a million sequences down to only 12,000. The previous bug in issue #311 resulted in uneven distributions when subsmpling these large samples with sub.sample but that is now fixed. However, when I run summary.shared on the original samples and set subsample=12000 I get very different numbers of OTUs for these highly subsampled samples. The greater the sub sampling effort, the greater the observed difference in OTUs between the sub.sample followed by summary.single and the summary.single with subsample=12000 methods. See the table below for the actual number of OTUS (sobs) returned by summary.single depending on whether I use all sequences without subsampling, pre subsample the shared file with sub.sample(nseqs=12000) followed by summary.single(), or just run summary.single on the original shared file with subsample=12000:

Sample	original sequence number	All sequences	sub.sample(nseqs=12000)	summary.shared(subsample=12000)
1	1,147,118	2,315	522	6.595
2	769,206	1,496	537	10.837
3	883,794	2,225	543	7.779
4	355,720	3,792	842	63.485
5	351,501	871	387	62.685
6	271,146	653	330	99.583
7	219,120	375	179	117.588
8	22,308	1,154	1,002	1,003.387
9	14,319	783	758	757.326
10	29,100	1,625	1,200	1,255.675
11	12,605	468	465	464.532
12	277,670	770	189	32.813
13	755,534	3,743	925	30.434
14	33,209	418	330	335.374
15	36,559	322	199	203.401

I am running the latest version of mothur 1.37.4 (including the commits that fix the sub.sample issue on windows). I am running 64-bit Windows 10.

Let me know if there is anything else I can provide to help with this issue.

Cheers
Richard

[mothur-westcott]

I found the source of the problem and have released version 1.39.5 which corrects it. https://github.com/mothur/mothur/releases/tag/v1.39.5

[campenr]

Thanks! Works great on my end now too 👍