Importing Nanopore reads to QIIME2

User Support
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Hello all,

I want to use QIIME 2 to analyse the nanopore reads I have for a total length of 16S. Since I already have V4 readings for the same samples and the analysis is available with epi2me, my goal is to gain higher resolution/coverage for the taxonomy of the taxa (I dont want to trim the nanopore reads).
The basecalling and adapter/barcode removal are already done.
My question is can I import these straight into Qiime2 or do I need to do any additional procedures, such utilising MetONTIIME, for example?
Also, is it necessary to account for the error rates (using Medaka for example)? Alternatively, should the QC score be lowered?
Could someone please provide me with a link to an instruction tutorial or advise me on where to start?

Maysa

2

Hi @maysa1,

Welcome back to the :qiime2: forum!

Have you checked out the user documentation?
There are tutorials in the documentation that you can follow and use as a guide for your analysis in qiime. A good place to start is the "Moving Pictures" tutorial.
I am not sure where you are in your analysis i.e what format your files are in, etc. so I cannot say if they are ready to be imported yet but if you search the forum there are plenty of people who have been in a similar situation and found solutions! That can be a great resource for you in getting started.
Let me know if you have any other questions after you check out those resources, I am happy to help :slight_smile:
--Hannah

2 Likes
3

Exactly!

Here is the command I used:

qiime tools import \
    --type 'SampleData[SequencesWithQuality]' \
    --input-path manifest.tsv \
    --input-format 'SingleEndFastqManifestPhred33V2' \
    --output-path $DATA/nanopore.qza

And manifest should contain 2 columns: 'sample-id 'and 'absolute-filepath'.

Best,

2 Likes
4

Thanks @jphagen and @timanix for your tips.

I appreciate your help, now I have a QIIME 2 artifact that does not look like what I would expect when working with a particular region (V4, V....). I have a few questions about the quality plots (the attached visualization file). Since 16S rRNA has a total length of 1500 bp, why am I seeing up to 6000 in my file? In addition, I am aware that denoising should result in a greater error rate for ON, the q2ONT pipeline used Vserch, can I use DADA2? If yes, how can I determine the truncation location given the quality plots?
nanopore-demux.qzv (437.2 KB)
I would be grateful for any feedback.

5

Hello!

I think, you need to discuss it with a person who performed library preparation and/or sequencing and/or initial processing of the output.

I would also recommend using VSEARCH since Dada2 is not appropriate for nanopore data.

Best,

1 Like
6

Thanks for replying, will use VSEARCH.

Maysa