# Problem with the start position after align.seqs **URL:** \ **Category:** Commands in mothur\ **Created:** [December 21, 2022, 4:18pm UTC](https://forum.mothur.org/t/problem-with-the-start-position-after-align-seqs/21672 "2022-12-21T16:18:36Z")\ **Posts on this page:** 5\ **Page:** 1
**Author:** ![etok](https://avatars.discourse-cdn.com/v4/letter/e/dc4da7/32.png) [@etok](https://forum.mothur.org/u/etok)\ **Post date:** [December 21, 2022, 4:18pm UTC](https://forum.mothur.org/t/problem-with-the-start-position-after-align-seqs/21672/1 "2022-12-21T16:18:36Z")
Hi, I’m new to using mothur and, actually, analyzing sequence data. As I’ve read a million times in this forum it is clearly a mistake to work with the V3-V4 region; unfortunately, it was too late to learn this when I got this data. So, I analyzed it by following the manual and forum, but, I have a problem with these ridiculously different start positions after alignment. This is the situation after **make.contigs:** ``` Start End NBases Ambigs Polymer NumSeqs ``` Minimum: 1 250 250 0 3 1 2.5%-tile: 1 274 274 0 4 143919 25%-tile: 1 298 298 0 4 1439188 Median: 1 442 442 0 5 2878375 75%-tile: 1 465 465 0 6 4317562 97.5%-tile: 1 466 466 2 31 5612831 Maximum: 1 500 500 75 250 5756749 Mean: 1 401.079 401.079 0.221197 7.30846 # of Seqs: 5756749 After that, I run **screen.seqs** : screen.seqs(fasta=stability.trim.contigs.fasta,minlength=10,maxlength=470,maxambig=0,maxhomop=8) ``` Start End NBases Ambigs Polymer NumSeqs ``` Minimum: 1 250 250 0 3 1 2.5%-tile: 1 274 274 0 4 125262 25%-tile: 1 298 298 0 4 1252620 Median: 1 441 441 0 5 2505240 75%-tile: 1 465 465 0 6 3757860 97.5%-tile: 1 466 466 0 6 4885218 Maximum: 1 470 470 0 8 5010479 Mean: 1 398.46 398.46 0 4.82017 # of Seqs: 5010479 Then, I run **unique.seqs** , create a count table, and run **align.seqs** with the tailored reference fasta that I produced from “silva.bacteria.fasta” by trimming it at the start position = 3388 and the end position= 25316. And, I got this: ``` Start End NBases Ambigs Polymer NumSeqs ``` Minimum: 0 0 0 0 1 1 2.5%-tile: 1 18929 7 0 2 125262 25%-tile: 1 18929 298 0 4 1252620 Median: 1 18929 441 0 5 2505240 75%-tile: 3979 18929 465 0 6 3757860 97.5%-tile: 8578 18929 466 0 6 4885218 Maximum: 18929 18929 470 0 8 5010479 Mean: 1874.55 18718 387.523 0 4.70551 # of unique seqs: 2887389 total # of seqs: 5010479 Well, as I understand it is not how it should be, and I do not know how I can fix this. What should I do? Thanks for all your time and help in advance. E.T. Update: I tried to set “minlength” parameter of **screen.seqs** to 390 bp and run it again. After, I followed the manual as I did before. The result I’ve got confused my mind even more. This is the summary of align.seqs: ``` Start End NBases Ambigs Polymer NumSeqs ``` Minimum: 1 14 3 0 1 1 2.5%-tile: 1 18928 439 0 4 80931 25%-tile: 1 18928 441 0 4 809302 Median: 1 18928 459 0 5 1618603 75%-tile: 1 18928 464 0 6 2427904 97.5%-tile: 1 18928 465 0 6 3156275 Maximum: 18910 18928 469 0 8 3237205 Mean: 26.8008 18798.8 450.293 0 4.92839 # of unique seqs: 2048792 total # of seqs: 3237205 As I search for it but I could not find any explanation. I used the same reference fasta as I used before. What does it mean? Thanks for your time and help again. E.T. ---
**Author:** ![leocadio](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/leocadio/32/203_2.png) [@leocadio](https://forum.mothur.org/u/leocadio)\ **Post date:** [December 22, 2022, 7:51pm UTC](https://forum.mothur.org/t/problem-with-the-start-position-after-align-seqs/21672/2 "2022-12-22T19:51:25Z")
For what you have here, your core reads are between 1 and 18928. All looks OK. Are you aligning to the whole 18S, or to a reference with just V3V4. It might help with the alignment… But, as you have it now, you have a bit over 3M reads, 2M unique, with start=1 and end=18928. Leo ---
**Author:** ![etok](https://avatars.discourse-cdn.com/v4/letter/e/dc4da7/32.png) [@etok](https://forum.mothur.org/u/etok)\ **Post date:** [December 22, 2022, 11:48pm UTC](https://forum.mothur.org/t/problem-with-the-start-position-after-align-seqs/21672/3 "2022-12-22T23:48:59Z")
Thanks for your reply. I am aligning illumina 16s v3v4 region data (screen.seqs was applied on the raw data with the parameters: maxhomop=8, maxambig=0, minlength=390 and maxlength= 470 to produce it) by using the reference fasta which I produced by trimming “silva.bacteria.fasta” file at start position=3388 and end position=25316 for the coverage 16s v3v4 region. I really do not know whether it is normal to get a start position “1” after alignment or if it is, what it means. I have strong feelings that I did something wrong because no other example is present to get a start position at “1” after align.seqs. Did I do something wrong if it is, what is it? Thanks for your time. ---
**Author:** ![etok](https://avatars.discourse-cdn.com/v4/letter/e/dc4da7/32.png) [@etok](https://forum.mothur.org/u/etok)\ **Post date:** [December 27, 2022, 11:58am UTC](https://forum.mothur.org/t/problem-with-the-start-position-after-align-seqs/21672/4 "2022-12-27T11:58:50Z")
After a week, I think I understand the situation. Sorry for taking your time. If I am correct, this is the story. The first problem I originally asked for help with was the start positions of the reads after alignment. Some of them were starting on “1” and some of them were starting on “3979” and “8578”. Suddenly, I realized that I was working with the 16s V3-V4 region, and the reads in the dataset are supposed to be ~ 460 bp. So, I run the screen.seqs command on the reads not only by the max length but also by the minlength and eliminated the reads shorter than 390 bp. When I aligned the sequences, all the sequences started at position “1”. The second question I was asking was related to the sequences starting at position “1”. Because this is the first time I am dealing with bioinformatics, and I had never seen the sequences that started at the position “1” in the tutorials and examples I found, I thought I had done something wrong. After days, I could finally think that it may be helpful to investigate the fastq files used for the examples and I saw that my sequences have primer sequences at the beginning and the sequences in the example fastq have not. So possibly they are aligning by beginning at the first position as they should be, there is no problem, just me being an anxious newbie. Thanks for your help. ---
**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:** [January 6, 2023, 11:59am UTC](https://forum.mothur.org/t/problem-with-the-start-position-after-align-seqs/21672/5 "2023-01-06T11:59:38Z")
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