# Pre.cluster removed all sequences **URL:** \ **Category:** Commands in mothur\ **Created:** [June 21, 2022, 6:25am UTC](https://forum.mothur.org/t/pre-cluster-removed-all-sequences/21459 "2022-06-21T06:25:34Z")\ **Posts on this page:** 4\ **Page:** 1
**Author:** ![SANTA-CATALINA](https://avatars.discourse-cdn.com/v4/letter/s/bb73d2/32.png) [@SANTA-CATALINA](https://forum.mothur.org/u/SANTA-CATALINA)\ **Post date:** [June 21, 2022, 6:25am UTC](https://forum.mothur.org/t/pre-cluster-removed-all-sequences/21459/1 "2022-06-21T06:25:34Z")
Hi everybody I work with mothur 1.48.0, when I run the pre.clusters command, all my sequences are removed. My script : ```auto make.contigs(file=fastq.files.table, oligos=miseq.LBE.16S.515_928.oligos.table, pdiffs=0, maxambig=0, maxhomop=8, processors=20) ## 220620 rename=T no function mothur 148 , moved up maxhomop=8 from screen.seqs below for consistency rename.seqs(fasta=current)## 220620 removed group=current, summary.seqs(fasta=current) unique.seqs(fasta=current) count.seqs(count=current) summary.seqs(count=current) align.seqs(fasta=current, reference=generic.LBE.16S.515_928.database.align) summary.seqs(fasta=current, count=current) screen.seqs(fasta=current, count=current, summary=current, start=180, end=13978) summary.seqs(fasta=current, count=current) filter.seqs(fasta=current, vertical=T, trump=.) unique.seqs(fasta=current, count=current) pre.cluster(fasta=fastq.files.trim.contigs.renamed.unique.good.filter.unique.fasta, count=fastq.files.trim.contigs.renamed.unique.good.filter.count_table, diffs=4, processors=1) ``` Logfile : ```auto mothur > make.contigs(file=fastq.files.table, oligos=miseq.LBE.16S.515_928.oligos.table, pdiffs=0, maxambig=0, maxhomop=8, processors=20) ## 220620 rename=T no function mothur 148 , moved up maxhomop=8 from screen.seqs below for consistency Using 20 processors. >>>>> Processing file pair ABwet_EN_1_24_05_OJ_ErT_GG_V4&GGACGG&L001&R1.fastq - ABwet_EN_1_24_05_OJ_ErT_GG_V4&GGACGG&L001&R2.fastq (files 1 of 2) <<<<< [WARNING]: your oligos file does not contain any group names. mothur will not create a groupfile. Making contigs... Done. It took 4 secs to assemble 50815 reads. >>>>> Processing file pair ABwet_En2_24_05_OJ_ErT_GG_V4&AGAGGG&L001&R1.fastq - ABwet_En2_24_05_OJ_ErT_GG_V4&AGAGGG&L001&R2.fastq (files 2 of 2) <<<<< [WARNING]: your oligos file does not contain any group names. mothur will not create a groupfile. Making contigs... Done. It took 4 secs to assemble 44531 reads. Group count: ABwet_EN_1_24_05_OJ_ErT_GG_V4 39119 ABwet_En2_24_05_OJ_ErT_GG_V4 33549 Total of all groups is 72668 It took 8 secs to process 95346 sequences. Output File Names: fastq.files.trim.contigs.fasta fastq.files.scrap.contigs.fasta fastq.files.contigs_report fastq.files.contigs.count_table mothur > rename.seqs(fasta=current)## 220620 removed group=current, Using fastq.files.trim.contigs.fasta as input file for the fasta parameter. Output File Names: fastq.files.trim.contigs.renamed.fasta fastq.files.trim.contigs.renamed_map mothur > summary.seqs(fasta=current) Using fastq.files.trim.contigs.renamed.fasta as input file for the fasta parameter. Using 20 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 308 308 0 3 1 2.5%-tile: 1 373 373 0 4 1817 25%-tile: 1 375 375 0 4 18168 Median: 1 376 376 0 5 36335 75%-tile: 1 377 377 0 5 54502 97.5%-tile: 1 377 377 0 6 70852 Maximum: 1 461 461 0 8 72668 Mean: 1 375 375 0 4 # of Seqs: 72668 It took 1 secs to summarize 72668 sequences. Output File Names: fastq.files.trim.contigs.renamed.summary mothur > unique.seqs(fasta=current) Using fastq.files.trim.contigs.renamed.fasta as input file for the fasta parameter. 72668 25475 Output File Names: fastq.files.trim.contigs.renamed.unique.fasta fastq.files.trim.contigs.renamed.count_table mothur > count.seqs(count=current) ##220620 removed name=current Using fastq.files.trim.contigs.renamed.count_table as input file for the count parameter. Output File Names: fastq.files.trim.contigs.renamed.sparse.count_table mothur > summary.seqs(count=current) Using fastq.files.trim.contigs.renamed.sparse.count_table as input file for the count parameter. Using fastq.files.trim.contigs.renamed.unique.fasta as input file for the fasta parameter. Using 20 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 308 308 0 3 1 2.5%-tile: 1 373 373 0 4 1817 25%-tile: 1 375 375 0 4 18168 Median: 1 376 376 0 5 36335 75%-tile: 1 377 377 0 5 54502 97.5%-tile: 1 377 377 0 6 70852 Maximum: 1 461 461 0 8 72668 Mean: 1 375 375 0 4 # of unique seqs: 25475 total # of seqs: 72668 It took 0 secs to summarize 72668 sequences. Output File Names: fastq.files.trim.contigs.renamed.unique.summary mothur > align.seqs(fasta=current, reference=generic.LBE.16S.515_928.database.align) Using fastq.files.trim.contigs.renamed.unique.fasta as input file for the fasta parameter. Unable to open generic.LBE.16S.515_928.database.align. Trying MOTHUR_FILES directory /mnt/sequencing/mothurdb/generic.LBE.16S.515_928.database.align. Using 20 processors. Reading in the /mnt/sequencing/mothurdb/generic.LBE.16S.515_928.database.align template sequences... DONE. It took 48 to read 213119 sequences. Aligning sequences from fastq.files.trim.contigs.renamed.unique.fasta ... It took 40 secs to align 25475 sequences. It took 41 seconds to align 25475 sequences. Output File Names: fastq.files.trim.contigs.renamed.unique.align fastq.files.trim.contigs.renamed.unique.align_report mothur > summary.seqs(fasta=current, count=current) Using fastq.files.trim.contigs.renamed.sparse.count_table as input file for the count parameter. Using fastq.files.trim.contigs.renamed.unique.align as input file for the fasta parameter. Using 20 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 173 5790 231 0 3 1 2.5%-tile: 180 13978 373 0 4 1817 25%-tile: 180 13978 375 0 4 18168 Median: 180 13978 376 0 5 36335 75%-tile: 180 13978 377 0 5 54502 97.5%-tile: 180 13978 377 0 6 70852 Maximum: 8110 14744 459 0 8 72668 Mean: 180 13976 375 0 4 # of unique seqs: 25475 total # of seqs: 72668 It took 0 secs to summarize 72668 sequences. Output File Names: fastq.files.trim.contigs.renamed.unique.summary mothur > screen.seqs(fasta=current, count=current, summary=current, start=180, end=13978) Using fastq.files.trim.contigs.renamed.sparse.count_table as input file for the count parameter. Using fastq.files.trim.contigs.renamed.unique.align as input file for the fasta parameter. Using fastq.files.trim.contigs.renamed.unique.summary as input file for the summary parameter. Using 20 processors. It took 2 secs to screen 25475 sequences, removed 92. / ****************************************** / Running command: remove.seqs(accnos=fastq.files.trim.contigs.renamed.unique.bad.accnos.temp, count=fastq.files.trim.contigs.renamed.sparse.count_table) Removed 118 sequences from fastq.files.trim.contigs.renamed.sparse.count_table. Output File Names: fastq.files.trim.contigs.renamed.sparse.pick.count_table / ****************************************** / Output File Names: fastq.files.trim.contigs.renamed.unique.good.summary fastq.files.trim.contigs.renamed.unique.good.align fastq.files.trim.contigs.renamed.unique.bad.accnos fastq.files.trim.contigs.renamed.sparse.good.count_table It took 2 secs to screen 25475 sequences. mothur > summary.seqs(fasta=current, count=current) Using fastq.files.trim.contigs.renamed.sparse.good.count_table as input file for the count parameter. Using fastq.files.trim.contigs.renamed.unique.good.align as input file for the fasta parameter. Using 20 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 173 13978 362 0 3 1 2.5%-tile: 180 13978 373 0 4 1814 25%-tile: 180 13978 375 0 4 18138 Median: 180 13978 376 0 5 36276 75%-tile: 180 13978 377 0 5 54413 97.5%-tile: 180 13978 377 0 6 70737 Maximum: 180 14744 381 0 8 72550 Mean: 179 13978 375 0 4 # of unique seqs: 25383 total # of seqs: 72550 It took 1 secs to summarize 72550 sequences. Output File Names: fastq.files.trim.contigs.renamed.unique.good.summary mothur > filter.seqs(fasta=current, vertical=T, trump=.) Using fastq.files.trim.contigs.renamed.unique.good.align as input file for the fasta parameter. Using 20 processors. Creating Filter... It took 1 secs to create filter for 25383 sequences. Running Filter... It took 0 secs to filter 25383 sequences. Length of filtered alignment: 542 Number of columns removed: 14202 Length of the original alignment: 14744 Number of sequences used to construct filter: 25383 Output File Names: fastq.filter fastq.files.trim.contigs.renamed.unique.good.filter.fasta mothur > unique.seqs(fasta=current, count=current) Using fastq.files.trim.contigs.renamed.sparse.good.count_table as input file for the count parameter. Using fastq.files.trim.contigs.renamed.unique.good.filter.fasta as input file for the fasta parameter. 25383 25383 Output File Names: fastq.files.trim.contigs.renamed.unique.good.filter.unique.fasta fastq.files.trim.contigs.renamed.unique.good.filter.count_table mothur > pre.cluster(fasta=fastq.files.trim.contigs.renamed.unique.good.filter.unique.fasta, count=fastq.files.trim.contigs.renamed.unique.good.filter.count_table, diffs=4, processors=1) Using 1 processors. 25383 3027 22356 Total number of sequences before precluster was 25383. pre.cluster removed 22356 sequences. / ****************************************** / [WARNING]: fastq.files.trim.contigs.renamed.unique.good.filter.unique.fasta does not contain any sequence from the .accnos file. Selected 0 sequences from fastq.files.trim.contigs.renamed.unique.good.filter.unique.fasta. Output File Names: fastq.files.trim.contigs.renamed.unique.good.filter.unique.precluster.fasta ``` I don’t understand why all the sequences are removed Thank you 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:** [June 21, 2022, 8:11pm UTC](https://forum.mothur.org/t/pre-cluster-removed-all-sequences/21459/2 "2022-06-21T20:11:52Z")
I’m not 100% what’s going on but your script is leaving out the count file at various places that it needs to be included. I’m surprised you aren’t getting other error messages earlier in the pipeline. 1.48.0 outputs a count file from `make.contigs` that you need to know which sequences belong to each group. You are leaving it out from your `rename.seqs` and `unique.seqs` commands. Maybe give this a try and see if it resolves the issues… ```auto make.contigs(file=fastq.files.table, oligos=miseq.LBE.16S.515_928.oligos.table, pdiffs=0, maxambig=0, maxhomop=8, processors=20) rename.seqs(fasta=current, count=current) summary.seqs(fasta=current, count=current) unique.seqs(fasta=current, count=current) summary.seqs(count=current) align.seqs(fasta=current, reference=generic.LBE.16S.515_928.database.align) summary.seqs(fasta=current, count=current) screen.seqs(fasta=current, count=current, summary=current, start=180, end=13978) summary.seqs(fasta=current, count=current) filter.seqs(fasta=current, vertical=T, trump=.) unique.seqs(fasta=current, count=current) pre.cluster(fasta=fastq.files.trim.contigs.renamed.unique.good.filter.unique.fasta, count=fastq.files.trim.contigs.renamed.unique.good.filter.count_table, diffs=4, processors=1) ``` ---
**Author:** ![SANTA-CATALINA](https://avatars.discourse-cdn.com/v4/letter/s/bb73d2/32.png) [@SANTA-CATALINA](https://forum.mothur.org/u/SANTA-CATALINA)\ **Post date:** [June 23, 2022, 10:23am UTC](https://forum.mothur.org/t/pre-cluster-removed-all-sequences/21459/3 "2022-06-23T10:23:55Z")
Thank you for your help. The problem is solved ---
**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:** [July 3, 2022, 10:24am UTC](https://forum.mothur.org/t/pre-cluster-removed-all-sequences/21459/4 "2022-07-03T10:24:44Z")
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