# Length of filtered alignment:0
**URL:** https://forum.mothur.org/t/length-of-filtered-alignment-0/20945
**Category:** Uncategorized
**Created:** [April 2, 2021, 11:00pm UTC](https://forum.mothur.org/t/length-of-filtered-alignment-0/20945 "2021-04-02T23:00:24Z")
**Posts on this page:** 7
**Page:** 1
### Author:  [@graciella](https://forum.mothur.org/u/graciella)
#### Post date: [April 2, 2021, 11:00pm UTC](https://forum.mothur.org/t/length-of-filtered-alignment-0/20945/1 "2021-04-02T23:00:24Z")
Hi,
I am using mothur v.1.45.1.
I trimmed the region of ecoli’s being amplified by the primers 341F and 805R. Aligning the trimmed sequence to the silva.bacteria.fasta, I got that the starting position is 6388 and the end position is 25316.
I used those values to run the pcr.seqs. Then, after running the filter.seqs, the output states that the length of the filtered alignment is 0.
I looked at other threads for the same topic, but I can not get what could cause this.
Thank you so much for any hint.
---
### Author:  [@pschloss](https://forum.mothur.org/u/pschloss)
#### Post date: [April 5, 2021, 5:37pm UTC](https://forum.mothur.org/t/length-of-filtered-alignment-0/20945/2 "2021-04-05T17:37:56Z")
Can you post the full commands you are running and their order?
Thanks,
Pat
---
### Author:  [@graciella](https://forum.mothur.org/u/graciella)
#### Post date: [April 5, 2021, 6:33pm UTC](https://forum.mothur.org/t/length-of-filtered-alignment-0/20945/3 "2021-04-05T18:33:14Z")
Thank you so much for your response. I appreciate it.
Please find below an extract of the log file with all the commands I used, until I got that the length of the filtered alignment was zero.
```auto
mothur >
make.file(inputdir=bottledwater, type=fastq, prefix=stability)
Setting input directory to: bottledwater\
mothur >
make.contigs(file=stability.files, processors=8)
mothur >
summary.seqs(fasta=stability.trim.contigs.fasta)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 298 298 0 3 1
2.5%-tile: 1 439 439 0 4 20288
25%-tile: 1 441 441 0 5 202872
Median: 1 464 464 0 5 405744
75%-tile: 1 465 465 1 6 608615
97.5%-tile: 1 466 466 10 6 791199
Maximum: 1 602 602 282 289 811486
Mean: 1 455 455 1 5
# of Seqs: 811486
mothur >
screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxambig=0, maxlength=466)
mothur >
get.current()
mothur >
summary.seqs()
Using bottledwater\stability.trim.contigs.good.fasta as input file for the fasta parameter.
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 301 301 0 3 1
2.5%-tile: 1 439 439 0 4 12559
25%-tile: 1 441 441 0 5 125585
Median: 1 464 464 0 5 251170
75%-tile: 1 465 465 0 6 376754
97.5%-tile: 1 465 465 0 6 489780
Maximum: 1 466 466 0 27 502338
Mean: 1 454 454 0 5
# of Seqs: 502338
mothur >
unique.seqs(fasta=stability.trim.contigs.good.fasta)
502338 401551
Output File Names:
bottledwater\stability.trim.contigs.good.names
bottledwater\stability.trim.contigs.good.unique.fasta
mothur >
count.seqs(name=stability.trim.contigs.good.names, group=stability.contigs.good.groups)
It took 8 secs to create a table for 502338 sequences.
Total number of sequences: 502338
Output File Names:
bottledwater\stability.trim.contigs.good.count_table
mothur >
summary.seqs(count=stability.trim.contigs.good.count_table)
Using bottledwater\stability.trim.contigs.good.unique.fasta as input file for the fasta parameter.
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 301 301 0 3 1
2.5%-tile: 1 439 439 0 4 12559
25%-tile: 1 441 441 0 5 125585
Median: 1 464 464 0 5 251170
75%-tile: 1 465 465 0 6 376754
97.5%-tile: 1 465 465 0 6 489780
Maximum: 1 466 466 0 27 502338
Mean: 1 454 454 0 5
# of unique seqs: 401551
total # of seqs: 502338
It took 7 secs to summarize 502338 sequences.
Output File Names:
bottledwater\stability.trim.contigs.good.unique.summary
mothur >
pcr.seqs(fasta=silva.bacteria.fasta, start=6388, end=25319, keepdots=F, processors=8)
Using 8 processors.
[NOTE]: no sequences were bad, removing bottledwater\silva.bacteria.bad.accnos
It took 12 secs to screen 14956 sequences.
Output File Names:
bottledwater\silva.bacteria.pcr.fasta
mothur >
rename.file(input=silva.bacteria.pcr.fasta, new=silva.v4.fasta)
mothur >
summary.seqs(fasta=silva.v4.fasta)
Unable to open bottledwater\silva.v4.fasta. Trying input directory bottledwater\silva.v4.fasta.
Unable to open bottledwater\silva.v4.fasta. Trying default C:\Users\gonzalg\Desktop\NEW_mothur\Mothur.win\mothur\silva.v4.fasta.
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 18930 421 0 3 1
2.5%-tile: 1 18931 441 0 4 374
25%-tile: 1 18931 444 0 4 3740
Median: 1 18931 463 0 5 7479
75%-tile: 1 18931 466 0 5 11218
97.5%-tile: 1 18931 467 1 6 14583
Maximum: 3 18931 508 5 9 14956
Mean: 1 18930 456 0 4
# of Seqs: 14956
It took 4 secs to summarize 14956 sequences.
Output File Names:
C:\Users\gonzalg\Desktop\NEW_mothur\Mothur.win\mothur\silva.v4.summary
mothur >
align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.v4.fasta)
Unable to open bottledwater\silva.v4.fasta. Trying input directory bottledwater\silva.v4.fasta.
Unable to open bottledwater\silva.v4.fasta. Trying default C:\Users\gonzalg\Desktop\NEW_mothur\Mothur.win\mothur\silva.v4.fasta.
Using 8 processors.
Reading in the C:\Users\gonzalg\Desktop\NEW_mothur\Mothur.win\mothur\silva.v4.fasta template sequences... DONE.
It took 6 to read 14956 sequences.
Aligning sequences from bottledwater\stability.trim.contigs.good.unique.fasta ...
It took 614 secs to align 401551 sequences.
[WARNING]: 401537 of your sequences generated alignments that eliminated too many bases, a list is provided in bottledwater\stability.trim.contigs.good.unique.flip.accnos.
[NOTE]: 401475 of your sequences were reversed to produce a better alignment.
It took 616 seconds to align 401551 sequences.
Output File Names:
bottledwater\stability.trim.contigs.good.unique.align
bottledwater\stability.trim.contigs.good.unique.align.report
bottledwater\stability.trim.contigs.good.unique.flip.accnos
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: 0 0 0 0 1 1
2.5%-tile: 1 18928 438 0 4 12559
25%-tile: 1 18928 440 0 5 125585
Median: 1 18928 463 0 5 251170
75%-tile: 1 18928 464 0 6 376754
97.5%-tile: 1 18928 464 0 6 489780
Maximum: 18931 18931 466 0 27 502338
Mean: 42 18926 453 0 5
# of unique seqs: 401551
total # of seqs: 502338
It took 148 secs to summarize 502338 sequences.
Output File Names:
bottledwater\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)
Using 8 processors.
It took 80 secs to screen 401551 sequences, removed 181.
/ ****************************************** /
Running command: remove.seqs(accnos=bottledwater\stability.trim.contigs.good.unique.bad.accnos.temp, count=bottledwater\stability.trim.contigs.good.count_table)
Removed 181 sequences from your count file.
Output File Names:
bottledwater\stability.trim.contigs.good.pick.count_table
/ ****************************************** /
Output File Names:
bottledwater\stability.trim.contigs.good.unique.good.summary
bottledwater\stability.trim.contigs.good.unique.good.align
bottledwater\stability.trim.contigs.good.unique.bad.accnos
bottledwater\stability.trim.contigs.good.good.count_table
It took 169 secs to screen 401551 sequences.
mothur >
summary.seqs(fasta=current, count=current)
Using bottledwater\stability.trim.contigs.good.good.count_table as input file for the count parameter.
Using bottledwater\stability.trim.contigs.good.unique.good.align as input file for the fasta parameter.
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 25 10 0 1 1
2.5%-tile: 1 18928 438 0 4 12554
25%-tile: 1 18928 440 0 5 125540
Median: 1 18928 463 0 5 251079
75%-tile: 1 18928 464 0 6 376618
97.5%-tile: 1 18928 464 0 6 489604
Maximum: 18899 18931 466 0 8 502157
Mean: 40 18927 453 0 5
# of unique seqs: 401370
total # of seqs: 502157
It took 140 secs to summarize 502157 sequences.
Output File Names:
bottledwater\stability.trim.contigs.good.unique.good.summary
mothur >
filter.seqs(fasta=stability.trim.contigs.good.unique.good.align, vertical=T, trump=.)
Using 8 processors.
Creating Filter...
It took 77 secs to create filter for 401370 sequences.
Running Filter...
It took 71 secs to filter 401370 sequences.
Length of filtered alignment: 0
Number of columns removed: 18931
Length of the original alignment: 18931
Number of sequences used to construct filter: 401370
Output File Names:
bottledwater\stability.filter
bottledwater\stability.trim.contigs.good.unique.good.filter.fasta
```
---
### Author:  [@pschloss](https://forum.mothur.org/u/pschloss)
#### Post date: [April 8, 2021, 5:58pm UTC](https://forum.mothur.org/t/length-of-filtered-alignment-0/20945/4 "2021-04-08T17:58:02Z")
Hi there,
In your second `screen.seqs` step…
```auto
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)
```
You need to include start and end coordinate positions. You might try something like this…
```auto
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=18928)
```
Also, it looks like you might be using 2x300 chemistry and are sequencing a longer region. You might want to check out this blog post from a few years back… [Why do I have such a large distance matrix](https://mothur.org/blog/2014/Why-such-a-large-distance-matrix/)
Pat
---
### Author:  [@graciella](https://forum.mothur.org/u/graciella)
#### Post date: [April 12, 2021, 1:53pm UTC](https://forum.mothur.org/t/length-of-filtered-alignment-0/20945/5 "2021-04-12T13:53:46Z")
Hi Pat,
Thank you so much.
The information in the link is relevant.
Including the start and end positions on the second screen.seqs step allowed to continue with: filter.seqs, unique.seqs and the pre.cluster commands.
However, at the chimera.vsearch, the program closed suddenly.
Thus, I got stuck.
This points to the difficulties to detect chimeras when using 2x300 chemistry as you mentioned in the link.
---
### Author:  [@pschloss](https://forum.mothur.org/u/pschloss)
#### Post date: [April 12, 2021, 8:51pm UTC](https://forum.mothur.org/t/length-of-filtered-alignment-0/20945/6 "2021-04-12T20:51:59Z")
Hi -
The chimera checking step shouldn’t crash out. Can you get the latest version of mothur and try again? It should be at [Release Version 1.45.2 · mothur/mothur · GitHub](https://github.com/mothur/mothur/releases/tag/v1.45.2). Let us know if it still crashes.
pat
---
### Author:  [@graciella](https://forum.mothur.org/u/graciella)
#### Post date: [April 20, 2021, 4:31pm UTC](https://forum.mothur.org/t/length-of-filtered-alignment-0/20945/7 "2021-04-20T16:31:28Z")
Hi
Version 1.45.2 is working great in analyzing my dataset !
The chimera check ran without problems.
I am now clustering the sequences into OTUs. I chose the traditional approach and it took about 24 h to get the distance for 233000 sequences. The clustering step has been running for 3 days…(on widows with 16 GB ram). It is taking long, but so far it looks fine.
Maybe at some point, I will compare it with the heuristic approach.
Thank you for your help !
Graciela