# Problems with Cluster.split **URL:** \ **Category:** Uncategorized\ **Created:** [July 18, 2022, 12:13am UTC](https://forum.mothur.org/t/problems-with-cluster-split/21479 "2022-07-18T00:13:18Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![AliceBaek](https://avatars.discourse-cdn.com/v4/letter/a/8491ac/32.png) [@AliceBaek](https://forum.mothur.org/u/AliceBaek)\ **Post date:** [July 18, 2022, 12:13am UTC](https://forum.mothur.org/t/problems-with-cluster-split/21479/1 "2022-07-18T00:13:18Z")
Hi, I am experiencing some difficulties getting past cluster.split in the MiSeq SOP. I sequenced the V4 region of the 16S rRNA gene on the MiSeq using the 2x250 kit. I followed the MiSeq SOP (mothur v.1.47.0) to make contigs and clean the dataset without any issues. I have tried a number of commands in an attempt to produce OTUs: ```auto make.file(inputdir=., type=gz, prefix=stability) make.contigs(file=stability.files, maxambig=0, maxlength=292, maxhomop=8, processors=24) summary.seqs(fasta=stability.trim.contigs.fasta, count=stability.contigs.count_table) Start End NBases Ambigs Polymer NumSeqs Minimum: 1 37 37 0 3 1 2.5%-tile: 1 291 291 0 3 190879 25%-tile: 1 291 291 0 4 1908783 Median: 1 292 292 0 4 3817565 75%-tile: 1 292 292 0 5 5726347 97.5%-tile: 1 292 292 0 5 7444251 Maximum: 1 300 300 0 8 7635129 Mean: 1 291 291 0 4 # of unique seqs: 7635129 total # of seqs: 7635129 It took 83 secs to summarize 7635129 sequences. mothur > unique.seqs(fasta=stability.trim.contigs.fasta, count=stability.contigs.count_table) mothur > summary.seqs(fasta=stability.trim.contigs.unique.fasta, count=stability.trim.contigs.count_table) Using 24 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 37 37 0 3 1 2.5%-tile: 1 291 291 0 3 190879 25%-tile: 1 291 291 0 4 1908783 Median: 1 292 292 0 4 3817565 75%-tile: 1 292 292 0 5 5726347 97.5%-tile: 1 292 292 0 5 7444251 Maximum: 1 300 300 0 8 7635129 Mean: 1 291 291 0 4 # of unique seqs: 2524391 total # of seqs: 7635129 mothur > align.seqs(fasta=stability.trim.contigs.unique.fasta, reference=/data/ref_database/mothur_silva/silva.nr_v138_1.align) summary.seqs(fasta=stability.trim.contigs.unique.align, count=stability.trim.contigs.count_table) Using 24 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1044 1048 1 0 1 1 2.5%-tile: 11895 25318 291 0 3 190879 25%-tile: 11895 25318 291 0 4 1908783 Median: 11895 25318 292 0 4 3817565 75%-tile: 11895 25318 292 0 5 5726347 97.5%-tile: 11895 25318 292 0 5 7444251 Maximum: 43116 43116 300 0 8 7635129 Mean: 11896 25318 291 0 4 # of unique seqs: 2524391 total # of seqs: 7635129 mothur > screen.seqs(fasta=stability.trim.contigs.unique.align, count=stability.trim.contigs.count_table, start=11895, end=25318) summary.seqs(fasta=stability.trim.contigs.unique.good.align, count=stability.trim.contigs.good.count_table) Using 24 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 10366 25318 260 0 3 1 2.5%-tile: 11895 25318 291 0 3 188773 25%-tile: 11895 25318 291 0 4 1887729 Median: 11895 25318 292 0 4 3775457 75%-tile: 11895 25318 292 0 5 5663185 97.5%-tile: 11895 25318 292 0 5 7362141 Maximum: 11895 25498 300 0 8 7550913 Mean: 11894 25318 291 0 4 # of unique seqs: 2466444 total # of seqs: 7550913 filter.seqs(fasta=stability.trim.contigs.unique.good.align, vertical=T, trump=.) unique.seqs(fasta=stability.trim.contigs.unique.good.filter.fasta, count=stability.trim.contigs.good.count_table) summary.seqs(fasta=stability.trim.contigs.unique.good.filter.unique.fasta, count=stability.trim.contigs.unique.good.filter.count_table) Using 24 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 611 260 0 3 1 2.5%-tile: 1 629 291 0 3 188773 25%-tile: 1 629 291 0 4 1887729 Median: 1 629 292 0 4 3775457 75%-tile: 1 629 292 0 5 5663185 97.5%-tile: 1 629 292 0 5 7362141 Maximum: 1 629 300 0 8 7550913 Mean: 1 628 291 0 4 # of unique seqs: 2447732 total # of seqs: 7550913 pre.cluster(fasta=stability.trim.contigs.unique.good.filter.unique.fasta, count=stability.trim.contigs.unique.good.filter.count_table, diffs=3) mothur > chimera.vsearch(fasta=stability.trim.contigs.unique.good.filter.unique.precluster.fasta, count=stability.trim.contigs.unique.good.filter.unique.precluster.count_table, dereplicate=t) mothur > summary.seqs(fasta=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.fasta, count=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.count_table) Using 24 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 611 260 0 3 1 2.5%-tile: 1 629 291 0 3 173851 25%-tile: 1 629 291 0 4 1738510 Median: 1 629 292 0 4 3477020 75%-tile: 1 629 292 0 5 5215530 97.5%-tile: 1 629 292 0 5 6780189 Maximum: 1 629 300 0 8 6954039 Mean: 1 628 291 0 4 # of unique seqs: 814566 total # of seqs: 6954039 classify.seqs(fasta=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.fasta, count=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.count_table, reference=/data/ref_database/mothur_silva/silva.nr_v138_1.align, taxonomy=/data/ref_database/mothur_silva/silva.nr_v138_1.tax) mothur > remove.lineage(fasta=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.fasta, count=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.count_table, taxonomy=stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.nr_v138_1.wang.taxonomy, taxon=Chloroplast-Mitochondria-unknown-Archaea-Eukaryota) mothur > rename.file(fasta=current, count=current, taxonomy=current, prefix=final) Using stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table as input file for the count parameter. Using stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.pick.fasta as input file for the fasta parameter. Using stability.trim.contigs.unique.good.filter.unique.precluster.denovo.vsearch.nr_v138_1.wang.pick.taxonomy as input file for the taxonomy parameter. ``` Next, I tried cluster.split several times in this way (1) mothur \> cluster.split(fasta=final.fasta, count=final.count\_table, taxonomy=final.taxonomy, taxlevel=4, processors=40) (2) mothur \> cluster.split(fasta=final.fasta, count=final.count\_table, taxonomy=final.taxonomy, taxlevel=4, processors=36) (3) mothur \> cluster.split(fasta=final.fasta, count=final.count\_table, taxonomy=final.taxonomy, taxlevel=5, processors=16) (4) mothur \> cluster.split(fasta=final.fasta, count=final.count\_table, taxonomy=final.taxonomy, taxlevel=6, processors=16) (5)mothur \> cluster.split(fasta=final.fasta, count=final.count\_table, taxonomy=final.taxonomy, taxlevel=6, processors=16, cutoff=0.10) (6)mothur \> cluster.split(fasta=final.fasta, count=final.count\_table, taxonomy=final.taxonomy, taxlevel=6, processors=8, cutoff=0.10) But, the result is: Out of memory: Killed process 2276 (mothur) total-vm: 263820292kB, anin-rss: 127037696kB, file-rss: 348kB, shmem-rss:0kB, UID: 1003 pgtables: 501792kB oom\_score\_adj:0 Killed How can I figure it out to get OTUs? (Total RAM: 128GB, processors:48) Thank you. ---
**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:** [July 18, 2022, 5:47pm UTC](https://forum.mothur.org/t/problems-with-cluster-split/21479/2 "2022-07-18T17:47:34Z")
Hi Alice, From the lengths of your contigs, I suspect your primers and barcodes are still attached to the sequences. You can use an oligos file in `make.contigs` to remove those sequences and assign the sequences to samples. Your V4 data should be about 252 nt long. Can you give that a try? If you still have barcodes on your sequences, then I suspect you have N-times as many unique sequences in this dataset than you really do (N = number of samples in dataset). Pat ---
**Author:** ![AliceBaek](https://avatars.discourse-cdn.com/v4/letter/a/8491ac/32.png) [@AliceBaek](https://forum.mothur.org/u/AliceBaek)\ **Post date:** [July 21, 2022, 8:16am UTC](https://forum.mothur.org/t/problems-with-cluster-split/21479/5 "2022-07-21T08:16:00Z")
Hi Pat, I thought carefully about your words. I sequenced the V4 region using the same 515F and 806R primers you used according to your Wet-lab SOP. Also, I confirmed that barcodes have been removed from the sequencing reads (sequencing company confirmed). I think that if we use 515F and 806R to generate PCR products before the sequencing, the length of PCR products should be 292 bp. And also think that if we make contigs with two paired reads, the length of contigs should be 292 nt (please refer to the figure I made below). But based on your reply and MiSeq SOP, the length of contigs are 252 nt. I do not fully understand why this happened. Could you let me know about this? Thanks for your help! Best, Alice ![16srRNA sequencing_V4 region](https://canada1.discourse-cdn.com/flex036/uploads/mothur/original/1X/9b4e96b99b9e41e2ff94b3b1c234a5a1dae090e1.png) ---
**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:** [July 21, 2022, 5:58pm UTC](https://forum.mothur.org/t/problems-with-cluster-split/21479/6 "2022-07-21T17:58:28Z")
Hi Alice, Can you post a few of your assembled sequences? I suspect they start with you forward primer. They will need to be removed to proceed with the rest of the analysis. Just because people use the same primers as us, that doesn’t mean that they are sequencing the same way that we recommend. Pat ---
**Author:** ![AliceBaek](https://avatars.discourse-cdn.com/v4/letter/a/8491ac/32.png) [@AliceBaek](https://forum.mothur.org/u/AliceBaek)\ **Post date:** [July 22, 2022, 1:21am UTC](https://forum.mothur.org/t/problems-with-cluster-split/21479/7 "2022-07-22T01:21:35Z")
Hi Pat, As you said, I saw the file created after make.contigs. As a result, there was a forward primer. However, I have a question here. I understand why the barcode should be removed, but I do not fully understand why the forward primer should be removed. Could you let me know about this? Also, what command should I use to remove forward primer? Thanks for your help! Best, Alice ![Part of stability.trim.contigs.fasta](https://canada1.discourse-cdn.com/flex036/uploads/mothur/original/1X/acc501724682e605c9e412c834bea0a309a83162.png) ---
**Author:** ![AliceBaek](https://avatars.discourse-cdn.com/v4/letter/a/8491ac/32.png) [@AliceBaek](https://forum.mothur.org/u/AliceBaek)\ **Post date:** [July 25, 2022, 2:35am UTC](https://forum.mothur.org/t/problems-with-cluster-split/21479/8 "2022-07-25T02:35:46Z")
Hi Pat, I made oligos file and removed the primer as you said. Then after make.contigs, the length of contigs came out to be 252 and I was able to solve the problem. Thank you so much for helping me solve the problem. Best, Alice ---
**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:** [August 2, 2022, 3:38pm UTC](https://forum.mothur.org/t/problems-with-cluster-split/21479/9 "2022-08-02T15:38:26Z")
Wonderful! Glad it’s working now. Pat