# Working out proceedure for nanopore 16s

**URL:** <https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154>\
**Category:** Uncategorized\
**Created:** [May 14, 2024, 5:56pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154 "2024-05-14T17:56:24Z")\
**Posts on this page:** 13\
**Page:** 1

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**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\
**Post date:** [May 14, 2024, 5:56pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/1 "2024-05-14T17:56:24Z")

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Creating this thread for my questions that arise when working through nanopore data because that could be useful for others. I’ll write up an SOP if the end result is worth others repeating.

Background. I’ve finally gotten my gridion and started sequencing. I have a few zymo mocks sequenced with nanopore’s 16s kit on R9.? (too high error to use mothur), 8f/1391R with nanopore PCR barcoded on R10.4, plus I’ve run them on MiSeq using standard v4 sequencing with EMP version of 515f/806R.

I’m trying to get the R10.4 data through mothur but I’m not super confident it’s going to give me anything useful. There are very few non-unique sequences which suggests too high error rates. This is still running (4 days and counting on 32 cores 500gb ram).

I’ve also pcr.seq selected out just v4 from this data and am trying to run mothur on that section to get a better idea of how this compares to illumina.

Finally, I’m going to repeat both these analyses on just the duplex data which is ~10% of the R10.4 run.

I’m presenting all this at ASM Microbe next month, so I have a deadline to get this done!

First few issues:  
trimming data: I’m using PCR seqs with pdiffs/rdiffs = 3 and 5

Here’s the batch that I’m trying to run. I’m sure that I’ll have to edit this as I find more errors. I’m on a high mem node, using 500gb

> mothur "#summary.seqs(fasta=oct23nanoporeRecall.fasta, processors=32);  
> pcr.seqs(fasta=current, count=oct23nanoporeRecall.count\_table, oligos=oligos8f1391r.txt, pdiffs=3, rdiffs=3, checkorient=T);  
> summary.seqs(fasta=current, count=current);  
> screen.seqs(fasta=current, maxambig=0, minlength=800, maxhomop=8);  
> unique.seqs(fasta=current);  
> summary.seqs(fasta=current, count=current);  
> align.seqs(fasta=current, reference=silva.nr\_v138\_1.align);  
> summary.seqs(fasta=current, count=current);  
> filter.seqs(fasta=current, vertical=T);  
> pre.cluster(fasta=current, count=current, diffs=14);  
> summary.seqs(fasta=current, count=current);  
> classify.seqs(fasta=current, count=current, reference=silva.nr\_v138\_1.align, taxonomy=silva.nr\_v138\_1.tax, cutoff=80);  
> remove.lineage(fasta=current, count=current, taxonomy=current, taxon=Chloroplast-Mitochondria-unknown-Eukaryota);  
> summary.tax(taxonomy=current, count=current);  
> dist.seqs(fasta=current, countends=F, cutoff= 0.03, processors=16);  
> cluster(column=current, count=current, method=opti);  
> summary.seqs(processors=32);  
> make.shared(list=current, count=current);  
> classify.otu(list=current, count=current, taxonomy=current);  
> get.oturep(fasta=current, count=current, list=current, method=abundance);  
> count.groups(shared=current);  
> rarefaction.single(shared=current);  
> summary.single(shared=current, calc=nseqs-sobs-coverage-shannon-shannoneven-invsimpson, subsample=10000);

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<div class="post-metadata">

**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:** [May 22, 2024, 12:28pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/2 "2024-05-22T12:28:12Z")

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Thanks for doing this Kendra. Since you’re at a core facility that has done both the Kozich protocol and nanopore sequencing, you’ll likely be able to answer this. Starting with genomic DNA, what do you estimate the cost to be to generate sequence data and how many reads of sequence data are being generated?

We were able to get very good PacBio full length sequence data that was as good as our Kozich protocol, but it would have been prohibitively expensive to do at the same scale. In my ranking quality is more important than cost, but cost is still important.

Thanks,  
Pat

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**Author:** ![Alexandre\_Thibodeau](https://avatars.discourse-cdn.com/v4/letter/a/e480ec/32.png) [@Alexandre\_Thibodeau](https://forum.mothur.org/u/Alexandre_Thibodeau)\
**Post date:** [May 27, 2024, 8:38pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/3 "2024-05-27T20:38:10Z")

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Quick question: how long does it take and on what type of computer? Can you also tell us how many samples you run per flow cell?

Thanks!

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<div class="post-metadata">

**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\
**Post date:** [May 28, 2024, 1:02pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/4 "2024-05-28T13:02:16Z")

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Cost is so dependent on throughput. If I can get enough data for 48 samples/flowcell, the cost will be very close to miseq which is my goal. I’m defining “enough data” as being at least a few thousand seqs as high quality as illumina since that’s what people expect. If I can’t get that, I’m going to wait till the next gen of flowcells/basecallers before launching the service.

We (royal we, the other UConn sequencing facility) now had a Sequal II that is very underutilized because they have a Revio. If nanopore really isn’t ready for amplicons, I’ll probably turn protocol dev towards pacbio. I just fundamentally want to get long reads to work on a $ sequencer rather than a $$$$$ sequencer.

As you know, one of the major hurdles in getting “enough” data for a bunch of samples is loading ~equal DNA for each barcode. I had an undergrad this past year working out a spri bead clean protocol for that. We make the PEG solution and dilute commercial beads with that to limit the amount of DNA bound. I have 96 nanopore barcodes ready to test with this method. hopefully this week.

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<div class="post-metadata">

**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\
**Post date:** [May 28, 2024, 1:10pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/5 "2024-05-28T13:10:41Z")

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@Alexandre_Thibodeau I’ve been playing with a test run that is 24 samples. I got ~8Gb but throw out 75% when matching primers. Those ~2.2 M reads never finished running using 32 cores and 500Gb ram. Dist.seqs (processors=2) ran for 2 weeks and I gave up (aka had other projects that I needed to get on that computer). Interestingly, I also trimmed out just v4 and it also ran for 2 weeks on dist.seqs and didn’t finish. I was able to run both the full length and trimmed v4 through dada2 but the results were nonsense.

I’ve pulled out just the duplex reads from this run ~0.5M reads. they ran through mothur overnight using 500gb ram. That just finished, I’ll look at the results today and see if they look more reasonable.

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<div class="post-metadata">

**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\
**Post date:** [June 14, 2024, 2:02pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/6 "2024-06-14T14:02:45Z")

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well boo. the duplex reads aren’t much better. I think this is going to require a deeper dive into the gnitty gritty of nanopore sequencing and basecalling.

If anyone’s at ASM, I’m presenting this tomorrow MBP-SATURDAY-651

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<div class="post-metadata">

**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\
**Post date:** [June 17, 2024, 9:15pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/7 "2024-06-17T21:15:47Z")

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Here’s my poster if you’re interested.

A number of ONT FAS came to talk to me, all insisting that there was a problem with my bioinformatics that was making the data look bad. I’m going to talk to some of them later this to see what the computational FAS think. They do have a new basecaller in the past month, so I’m trying to get that rerun.Though honestly, I’m going to be worried if it changes the calls so much that it drops the error rate significantly.

@pschloss I ran seq.error but don’t know how to use the output beyond that one high level overall rate. Do you have scripts for what to do with it? Here’s the report for in silico v4 (pulled from the full sequences with pcr.seqs)

> Errors Sequences  
> 0 787266  
> 1 3872  
> 2 568  
> 3 47  
> 4 14  
> 5 8  
> 6 4  
> 7 1

And here’s the summary for the full length

> Errors Sequences  
> 0 20336  
> 1 4031  
> 2 459  
> 3 18  
> 4 16  
> 5 31  
> 6 0  
> 7 0  
> 8 4  
> 9 1  
> 10 0  
> 11 0  
> 12 0  
> 13 3  
> 14 0  
> 15 0  
> 16 0  
> 17 0  
> 18 0  
> 19 0  
> 20 1  
> 21 0  
> 22 0  
> 23 1

> **[20240612ASM.pdf](https://mars.media.uconn.edu/wp-content/uploads/sites/1260/2024/06/20240612ASM.pdf)**
>
> 1772.48 KB

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<div class="post-metadata">

**Author:** ![Kendra](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/kendra/32/112_2.png) [@Kendra](https://forum.mothur.org/u/Kendra)\
**Post date:** [June 24, 2024, 6:55pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/8 "2024-06-24T18:55:06Z")

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@graciella here’s what I’m trying

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**Author:** ![Martin](https://avatars.discourse-cdn.com/v4/letter/m/73ab20/32.png) [@Martin](https://forum.mothur.org/u/Martin)\
**Post date:** [November 20, 2024, 12:32pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/9 "2024-11-20T12:32:48Z")

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Hello, I followed MiseqSOP last year with our illumina data but last week I got some nanopore 16s data and wanted to analyze it with mothur. I wanted to follow your procedure but I have maybe kinda noobish question - how can I generate count\_table? cus with MiseqSOP it is created with make.contigs, but I can´t do it with nanopore single read data. Also to create fasta from my fastq files can I just use fastq.info with pacbio=T as I don´t have forward and reverse read? or it is suitable only for pacbio data and not nanopore?

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<div class="post-metadata">

**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 20, 2024, 2:54pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/10 "2024-11-20T14:54:14Z")

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You can get a count file using [count.seqs](https://mothur.org/wiki/count.seqs/#group) with a names and group file

Pat

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<div class="post-metadata">

**Author:** ![Martin](https://avatars.discourse-cdn.com/v4/letter/m/73ab20/32.png) [@Martin](https://forum.mothur.org/u/Martin)\
**Post date:** [November 21, 2024, 9:31am UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/11 "2024-11-21T09:31:41Z")

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Thank you for your reply Pat! How can I get group or names file or how they should be formated? All I have now are nanopore trimmed fastq files for each barcode.

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<div class="post-metadata">

**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, 2024, 2:00pm UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/12 "2024-11-21T14:00:06Z")

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You can get a names file from running unique.seqs - this will also generate a count file for you. You’ll have to make your own group file. It’s a tab-separated values (TSV) file with the first column having the sequence name and the second is the group it belongs to.

Pat

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**Author:** ![dporco](https://avatars.discourse-cdn.com/v4/letter/d/bcef8e/32.png) [@dporco](https://forum.mothur.org/u/dporco)\
**Post date:** [October 2, 2026, 7:09am UTC](https://forum.mothur.org/t/working-out-proceedure-for-nanopore-16s/22154/13 "2026-10-02T07:09:01Z")

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Hello Kendra

Sorry for digging out this old topic. I am trying some R10.4 nifh sequencing (around 450bp) called with Dorado SUP accuracy, looking for info.

You preclustered data at diffs=14 for V4 515f/806R (350bp), why using such a high value? Did you test several ones to find this one was the best compromise ?

I saw your poster that looks like a funeral for the platform. Did you make some progress on this or was it out of recovery ?

Thanks

David