# Protocol for dealing with 16s full-length sequencing data by Pacbio **URL:** \ **Category:** Uncategorized\ **Created:** [November 13, 2022, 7:00am UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622 "2022-11-13T07:00:40Z")\ **Posts on this page:** 9\ **Page:** 1
**Author:** ![hgvxin](https://avatars.discourse-cdn.com/v4/letter/h/4491bb/32.png) [@hgvxin](https://forum.mothur.org/u/hgvxin)\ **Post date:** [November 13, 2022, 7:00am UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/1 "2022-11-13T07:00:40Z")
Dear all Do you have a protocol to deal with the Pacbio sequencing data using mothur. ---
**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 21, 2022, 3:58pm UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/2 "2022-11-21T15:58:36Z")
I don’t have a web-based SOP, but you can read through my paper to get an idea of what to do… > **[Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system](https://peerj.com/articles/1869/)** > > Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq,... Pat ---
**Author:** ![jugsy67](https://avatars.discourse-cdn.com/v4/letter/j/6f9a4e/32.png) [@jugsy67](https://forum.mothur.org/u/jugsy67)\ **Post date:** [November 25, 2022, 1:59pm UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/3 "2022-11-25T13:59:30Z")
Hi Pat so you think that this SOP will deal with the near full length PacBio HiFi reads we are getting? cheers Julian ---
**Author:** ![jugsy67](https://avatars.discourse-cdn.com/v4/letter/j/6f9a4e/32.png) [@jugsy67](https://forum.mothur.org/u/jugsy67)\ **Post date:** [November 25, 2022, 2:02pm UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/4 "2022-11-25T14:02:38Z")
ps would you be willing to share the logfile? ---
**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 28, 2022, 9:48pm UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/5 "2022-11-28T21:48:12Z")
Here is the entire codebase for that paper: > **[GitHub - SchlossLab/Schloss\_PacBio16S\_PeerJ\_2016: Repo for Sequencing 16S...](https://github.com/SchlossLab/Schloss_PacBio16S_PeerJ_2016)** > > Repo for Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system - GitHub - SchlossLab/Schloss\_PacBio16S\_PeerJ\_2016: Repo for Sequencing 16S rRNA gene fragments using the Pac... ---
**Author:** ![hgvxin](https://avatars.discourse-cdn.com/v4/letter/h/4491bb/32.png) [@hgvxin](https://forum.mothur.org/u/hgvxin)\ **Post date:** [November 29, 2022, 5:55am UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/6 "2022-11-29T05:55:50Z")
well getting your file, thanks so much 😁 ---
**Author:** ![jugsy67](https://avatars.discourse-cdn.com/v4/letter/j/6f9a4e/32.png) [@jugsy67](https://forum.mothur.org/u/jugsy67)\ **Post date:** [November 29, 2022, 1:37pm UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/7 "2022-11-29T13:37:37Z")
sorry to be dumb, but i looked at the github page and can’t find the mothur commands for running PabBio seqs ---
**Author:** ![jugsy67](https://avatars.discourse-cdn.com/v4/letter/j/6f9a4e/32.png) [@jugsy67](https://forum.mothur.org/u/jugsy67)\ **Post date:** [November 30, 2022, 5:51pm UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/8 "2022-11-30T17:51:12Z")
I tried running Mothur using these cmds fastq.info(file=PB\_16S\_group.txt, pacbio=T) summary.seqs(fasta=PB\_16S\_group.fasta, count=PB\_16S\_group.count\_table, processors=6) screen.seqs(fasta=PB\_16S\_group.fasta, count=PB\_16S\_group.count\_table, maxambig=0, maxlength=1493, maxhomop=8) summary.seqs(fasta=current) unique.seqs(fasta=current) summary.seqs(fasta=current, count=current) align.seqs(fasta=current, reference=silva.nr\_v138\_1.align, processors=10) summary.seqs(fasta=current, count=current) screen.seqs(fasta=current, count=current, start=1044, end=43116) summary.seqs(fasta=current, count=current) filter.seqs(fasta=current, vertical=T, trump=.) and then it crashed with this error msg Using PB\_16S\_group.good.unique.good.good.align as input file for the fasta parameter. Using 8 processors. Creating Filter… [ERROR]: Sequences are not all the same length, please correct. It took 63 secs to create filter for 202895 sequences. These files work in DADA2 but I want to also test an good old fashion 97% OTU approach - any help welcome ---
**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:** [December 1, 2022, 10:16pm UTC](https://forum.mothur.org/t/protocol-for-dealing-with-16s-full-length-sequencing-data-by-pacbio/21622/9 "2022-12-01T22:16:41Z")
It looks like you have too many "good"s in the file name for the pipeline you gave. At what command did it start complaining? You might go back to where you don’t get an error message and then step through each of the commands Pat