# pcr.seqs bug? in v.1.33.3 **URL:** \ **Category:** mothur bugs\ **Created:** [November 5, 2014, 12:17am UTC](https://forum.mothur.org/t/pcr-seqs-bug-in-v-1-33-3/2114 "2014-11-05T00:17:44Z")\ **Posts on this page:** 3\ **Page:** 1
**Author:** ![manureva](https://avatars.discourse-cdn.com/v4/letter/m/f475e1/32.png) [@manureva](https://forum.mothur.org/u/manureva)\ **Post date:** [November 5, 2014, 12:17am UTC](https://forum.mothur.org/t/pcr-seqs-bug-in-v-1-33-3/2114/1 "2014-11-05T00:17:44Z")
Hi, I’m new to Mothur and I’m therefore using the MiSeqSOP with the tutorial’s dataset to get familiar with the pipeline before playing with my own data. I think I might have spotted a bug on Mothur v.1.33.3 (Windows 32-bit version). Well, at least there’s something that bugs me…In order to follow the SOP I downloaded the silva.seed\_v119.align database on the 3rd of November 2014 (2 days ago). The problem appears when I run the following command: pcr.seqs(fasta=silva.seed\_v119.align, start=11894, end=25319, keepdots=F, processors=1) system(rename=silva.seed\_v119.pcr.align silva.v4.fasta) summary.seqs(fasta=silva.v4.fasta) Instead of getting this, as the SOP states: Start End NBases Ambigs Polymer NumSeqs Minimum: 1 13424 270 0 3 1 2.5%-tile: 1 13425 292 0 4 374 25%-tile: 1 13425 293 0 4 3740 Median: 1 13425 293 0 4 7479 75%-tile: 1 13425 293 0 5 11218 97.5%-tile: 1 13425 294 1 6 14583 Maximum: 3 13425 351 5 9 14956 Mean: 1.00074 13425 292.977 0.0573014 4.57014 # of Seqs: 14956 I get this: Start End Nbases Ambigs Polymer NumSeqs Minimum: 1 1150 260 0 3 1 2.5%-tile: 1 13425 292 0 4 376 25%-tile: 1 13425 293 0 4 3753 Median: 1 13425 293 0 5 7505 75%-tile: 1 13425 293 0 5 11257 97.5%-tile: 1 13425 460 1 6 14634 Maximum: 3 13425 1122 5 9 15009 Mean: 1.0006 13424.9 321.419 0.064428 1.78433 # of Seqs: 15009 I understand that perhaps the database got 53 sequences bigger between the time you wrote the SOP and the day I downloaded it. So I got rid of the sequences (in theory the 53 additional sequences because 15009-14956=43) that were smaller than 270 and longer than 351 in order to match the SOP output. To do so I ran: trim.seqs(fasta=silva.v4.fasta, minlength=270, maxlength=351, processors=1) summary.seqs(fasta=silva.v4.trim.fasta) which gives me this: Start End Nbases Ambigs Polymer NumSeqs Minimum: 1 1150 270 0 3 1 2.5%-tile: 1 13425 292 0 4 312 25%-tile: 1 13425 293 0 4 3114 Median: 1 13425 293 0 4 6227 75%-tile: 1 13425 293 0 5 9340 97.5%-tile: 1 13425 294 1 6 12141 Maximum: 1 13425 351 5 9 12452 Mean: 1 293.056 293.056 0.061115 4.5983 # of Seqs: 12452 So it turns out if I get rid of the sequences that in theory make up for the difference between my results and the ones from the SOP I end up getting rid of 2557 sequences (15009 originally, but only 12452 left after trim.seqs), instead of the theoretical 53 (15009 sequence in my silva.seed\_v119.align but only 14956 in yours). Questions are: 1. How can that be? 2. Should I be worried about using this DB, is this a real bug or something I can ignore? Many thanks in advance for your help! ---
**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:** [November 5, 2014, 10:12pm UTC](https://forum.mothur.org/t/pcr-seqs-bug-in-v-1-33-3/2114/2 "2014-11-05T22:12:36Z")
Hi the seed in the SOP is the older version of the seed that only had 14956 sequences. The newer one has more sequences and was actually created in a slightly different manner. You should be ok. Pat ---
**Author:** ![manureva](https://avatars.discourse-cdn.com/v4/letter/m/f475e1/32.png) [@manureva](https://forum.mothur.org/u/manureva)\ **Post date:** [November 5, 2014, 10:40pm UTC](https://forum.mothur.org/t/pcr-seqs-bug-in-v-1-33-3/2114/3 "2014-11-05T22:40:38Z")
OK, thanks!