# Screen.seq fail

**URL:** <https://forum.mothur.org/t/screen-seq-fail/22199>\
**Category:** Commands in mothur\
**Created:** [August 30, 2024, 4:07pm UTC](https://forum.mothur.org/t/screen-seq-fail/22199 "2024-08-30T16:07:03Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![JuanFrene](https://avatars.discourse-cdn.com/v4/letter/j/eb9ed0/32.png) [@JuanFrene](https://forum.mothur.org/u/JuanFrene)\
**Post date:** [August 30, 2024, 4:07pm UTC](https://forum.mothur.org/t/screen-seq-fail/22199/1 "2024-08-30T16:07:03Z")

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I am having difficulties figuring out why screen.seqs is completely removing \>99% of my reads. As per the SOP, you run screen.seqs to get sequences that start at or before/after the start/end positions. However, instead of keeping these sequences, it completely eliminates them.

I am using mothur v.1.48.1

```auto
mothur > 
summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table)

Using 8 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	1	1	0	1	1
2.5%-tile:	1	18929	440	0	4	95676
25%-tile:	1	18929	442	0	5	956752
Median: 1	18929	460	0	5	1913504
75%-tile:	1	18929	465	0	6	2870255
97.5%-tile:	1	18929	466	0	8	3731331
Maximum:	18932	18932	466	0	8	3827006
Mean:	19	18914	454	0	5
# of unique seqs:	2695221
total # of seqs:	3827006

It took 1513 secs to summarize 3827006 sequences.

Output File Names:
stability.trim.contigs.good.unique.summary

mothur > 
screen.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, summary=stability.trim.contigs.good.unique.summary, maxhomop=8, start=1, end=18932)

Using 8 processors.

It took 1125 secs to screen 2695221 sequences, removed 2694249.

/ ****************************************** /
Running command: remove.seqs(accnos=stability.trim.contigs.good.unique.bad.accnos.temp, count=stability.trim.contigs.good.count_table)
Removed 3825869 sequences from stability.trim.contigs.good.count_table.

Output File Names:
stability.trim.contigs.good.pick.count_table

/ ****************************************** /

Output File Names:
stability.trim.contigs.good.unique.good.summary
stability.trim.contigs.good.unique.good.align
stability.trim.contigs.good.unique.bad.accnos
stability.trim.contigs.good.good.count_table

It took 1596 secs to screen 2695221 sequences.

```

Thank you so much for your help

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<div class="post-metadata">

**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 3, 2024, 1:58pm UTC](https://forum.mothur.org/t/screen-seq-fail/22199/2 "2024-09-03T13:58:40Z")

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Thanks for your question. `screen.seqs` is doing exactly what you told it to 🤣

You used `start=1, end=18932` as arguments. This means that each sequence that passes should start at or before 1 and end at or after 18932. I suspect nearly all of your sequences end at 18929, which of course, is before 18932 so those sequences are getting removed. I’d suggest using `start=1, end=18929`.

Also, it appears you’re using one of the longer regions of the 16S rRNA genes. I’d encourage you to check out this blogpost from a few years ago that is still painfully relevant…

> **[Why do I have such a large distance matrix](https://mothur.org/blog/2014/Why-such-a-large-distance-matrix/)**
>
> The website that supports the mothur software program - one of the most widely used tools for analyzing 16S rRNA gene sequence data. Step inside to learn how to use the software, get help, and join our community!

Take care,  
Pat

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<div class="post-metadata">

**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\
**Post date:** [September 13, 2024, 1:58pm UTC](https://forum.mothur.org/t/screen-seq-fail/22199/3 "2024-09-13T13:58:56Z")

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