Merging paired end sequences after importing fastq manifest data

Hello,

I am very new to Qiime2 and I successfully imported my sequence data via paired-end fastqmanifestphred33 format. I think I am getting confused on the step to take after this importing step. I was able to run through the moving pictures tutorial with the sample data however applying my own data to those commands is tripping me up. The sequences are already demultiplexed so I tried skipping over that step in the tutorial to generate a feature table but found that to not work. I then tried running the demultiplexing step anyway for paired-end sequences in the Atacama soil tutorial and the error below popped up. This is leading me to think that my sequences were merged incorrectly into qiime2 so I am a little lost on what step to take.

Sorry for the long question, I just need a very basic answer on if my import worked and then the next step to take after I received a paired-end-demux.qza output

Plugin error from demux:

** Argument to input ‘seqs’ is not a subtype of EMPPairedEndSequences.**

Debug info has been saved to /var/folders/m6/kby0dlrd3cz8gsrdfqchxg680000gn/T/qiime2-q2cli-err-vpa94vfb.log.

Thank you so much for any help!

Hi @twhitney, can you provide the following things to help us with your request:

  • The exact command you used to import your data
  • A sample of your manifest file (just the first 5 lines or so)

Can you provide some more detail here about how it didn’t work? What commands did you run? What errors did you see? Please attach any relevant error logs that QIIME 2 produces.

I think this makes sense — if the data was already demultiplexed, attempting to demultiplex it again isn’t going to work!

Thanks! :sunny:

The command for importing was the one below and it ran through with no errors.

(qiime2-2017.6) 128-223-223-233-wireless:~ TWhitney$ qiime tools import --type SampleData[PairedEndSequencesWithQuality] --input-path RC16s.csv --output-path paired-end-demuxRCs.qza --source-format PairedEndFastqManifestPhred33

I am now confused with the next steps to take because

  1. It talks about a metadata table but when I ran through the moving-pcitures-tutorial before, it wasn’t necessary to have a metadata prior to the run

  2. If I don’t need to demultiplex steps (which I don’t) how do I get to the feature table command without the outputs from the demultiplexing and sequence control steps?

The command I ran for the plug-in error above is

(qiime2-2017.6) 128-223-223-233-wireless:~ TWhitney$ qiime demux emp-paired --m-barcodes-file sample-metadata.tsv --m-barcodes-category BarcodeSequence --i-seqs paired-end-demuxRCs.qza --o-per-sample-sequences demux --p-rev-comp-mapping-barcodes

Having the --m-barcodes-file sample-metadata.tsv is confusing me, do I need to create my own metadata table to run any of the tutorials?

For wanting a sample of my manifest file… if it is saved as a .qza file how would that work to view in the q2 studio?

Sorry I know this is a lot and not sure if I provided the correct information needed to get to the answer.

Thanks for the reply!

Hi @twhitney:

Your manifest file is the CSV you specified during your import step, so judging from the command you just provided, that filename is called RC16s.csv. You can attach that file, or paste just the first 5-10 lines of the file’s contents.

Since your data is already demultiplexed, the next step you should take after importing is to proceed straight to quality control, using either q2-dada2 or q2-deblur! Using q2-dada2, you would pick up in the Atacama tutorial here, specifically at the qiime demux summarize step. You will need to specify the filename of your demultiplexed sequences in the summarize command, so it will look something like this:

$ qiime demux summarize \
  --i-data paired-end-demuxRCs.qza \
  --o-visualization demux.qzv

Then, you can use that summarize visualization to assist with determining trimming and/or truncation parameters to use in the next step, dada2 denoise paired:

qiime dada2 denoise-paired \
  --i-demultiplexed-seqs paired-end-demuxRCs.qza \
  --o-table table.qza \
  --o-representative-sequences rep-seqs.qza \
  --p-trim-left-f X1 \
  --p-trim-left-r X2 \
  --p-trunc-len-f Y1 \
  --p-trunc-len-r Y2

Where X1, X2, Y1, and Y2 are all parameters that you determine based on your data (using the demux summarize visualization to assist).

Once denoised, then you can proceed with the rest of the Moving Pictures tutorial, for example picking up here.

Let us know if you need any help!

Thank you so much for the helpful response the summary plot turned out!

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