# Short sequences following screen.seqs and filter.seqs **URL:** \ **Category:** Commands in mothur\ **Created:** [October 16, 2012, 11:46pm UTC](https://forum.mothur.org/t/short-sequences-following-screen-seqs-and-filter-seqs/1087 "2012-10-16T23:46:54Z")\ **Posts on this page:** 4\ **Page:** 1
**Author:** ![skeletor](https://avatars.discourse-cdn.com/v4/letter/s/ac91a4/32.png) [@skeletor](https://forum.mothur.org/u/skeletor)\ **Post date:** [October 16, 2012, 11:46pm UTC](https://forum.mothur.org/t/short-sequences-following-screen-seqs-and-filter-seqs/1087/1 "2012-10-16T23:46:54Z")
Hello all, I have a couple of 16S datasets obtained from gut biopsies that I am trying to analyze. However, in both datasets I am getting really poor classification past the phylum level. I’ve noticed that following the screening/filtering steps after the alignment, I end up with really short sequences (from 186bp before the alignment to 67bp after filtering). I have tried playing around with the screen.seqs parameters, and have also tried aligning to the GreenGenes database, but I keep getting the same results. I have the same problem with both datasets. For example, here is a summary of what I have following the alignment step in one of my datasets: Start End NBases Ambigs Polymer NumSeqs Minimum: 1044 1053 5 0 2 1 2.5%-tile: 1044 1213 55 0 3 1728 25%-tile: 1044 2532 123 0 4 17272 Median: 1044 4978 197 0 4 34544 75%-tile: 1044 5413 246 0 5 51815 97.5%-tile: 1044 6409 322 0 6 67359 Maximum: 43111 43116 360 0 7 69086 Mean: 1077.25 4201.27 186.622 0 4.268 # of Seqs: 69086 After running: screen.seqs(fasta=calgary.trim.unique.align,name=calgary.trim.names,group=calgary.groups,start=1044,optimize=end,criteria=85) filter.seqs(fasta=calgary.trim.unique.good.align,vertical=T,trump=.,processors=12) Start End NBases Ambigs Polymer NumSeqs Minimum: 1 237 45 0 2 1 2.5%-tile: 1 237 52 0 2 1565 25%-tile: 1 237 65 0 3 15648 Median: 1 237 68 0 4 31296 75%-tile: 1 237 71 0 4 46944 97.5%-tile: 1 237 75 0 4 61027 Maximum: 1 237 108 0 6 62591 Mean: 1 237 67.2747 0 3.46726 # of Seqs: 62591 Does anyone have any ideas what could be going wrong here? Thanks in advance! Kyle ---
**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:** [October 17, 2012, 1:37pm UTC](https://forum.mothur.org/t/short-sequences-following-screen-seqs-and-filter-seqs/1087/2 "2012-10-17T13:37:40Z")
So you have the start position right. The problem is that your sequences are generally pretty short - 25% of your reads are less than 123 bases. You might need to change criteria to equal 50. You probably also want to ask your sequencing facility why the reads were so bad. As you probably have guessed, short reads will not classify very well. Pat ---
**Author:** ![skeletor](https://avatars.discourse-cdn.com/v4/letter/s/ac91a4/32.png) [@skeletor](https://forum.mothur.org/u/skeletor)\ **Post date:** [October 17, 2012, 4:58pm UTC](https://forum.mothur.org/t/short-sequences-following-screen-seqs-and-filter-seqs/1087/3 "2012-10-17T16:58:10Z")
Thanks Pat, I appreciate your input. I was hoping it was something that I was doing wrong, rather than a data quality issue. Kyle ---
**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\ **Post date:** [October 18, 2012, 11:07pm UTC](https://forum.mothur.org/t/short-sequences-following-screen-seqs-and-filter-seqs/1087/4 "2012-10-18T23:07:27Z")
from the filter.seqs page > NOTE: having one or two sequences included that don’t align with the bulk of your sequences may lead to all columns being removed by the trump option! I often have a few bad seqs slip through to this step (like your maximal sequence) if you use trump=. in that case you’ll throw away a lot of good bases because they don’t overlap with the few bad seqs