Next steps following Importing demultiplexed sequences data in Casava format

I am new to use QIIME2 after the QIIME workshop. I have imported our paired-end demultiplex data into QIIME2 following the Casava Format. I am trying to explore how to get the next analysis steps so that I can following analysis as in the tutorial sections? Do I need to join the paired-end sequence into one sequence or any other pre-processing steps before following the tutorial steps? Please guide me and thank you so much!

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Hi @Bing,
It sounds like you should now be ready to follow the workflow presented in the Moving Pictures tutorial. qiime dada2 denoise-paired will join the paired-end reads for you. Let us know if you run into any issues!

Hi @gregcaporaso
Thank you for your immediate response. I have created the demux-paired-end.qza. Then I go to “Moving picture tutorial” and jumped over “the demultiplexing sequence”, and directly go to “Sequence quality control”, is that right? I got the errors. See below and please guide me:

(qiime2-2017.2) Jinbings-iMac:qiime2 jinbingbai$ ls
casava-18-paired-end-demultiplexed	untitled folder
demux-paired-end.qza
(qiime2-2017.2) Jinbings-iMac:qiime2 jinbingbai$ qiime dada2 plot-qualities \
> --i-demultiplexed-seqs demux-paired-end.qza \
> --p-n 10 \
> --o-visualization demux-paired-end-qual-pilots.qzv
    Plugin error from dada2:

      Command '['profile_quality.R',
      '/var/folders/sd/vlrr9d916gg5qj3yzzf3srfw0000gn/T/qiime2-archive-esbp6
      kpb/f87e89c9-0ca1-44b6-b4fe-a0ec8f441cfb/data/DBR21181_GYN6015Visit1_P
      re_RTAliquot_1_S5_L001_R1_001.fastq.gz', '/var/folders/sd/vlrr9d916gg5
      qj3yzzf3srfw0000gn/T/qiime2-temp-a77wcy73']' returned non-zero exit
      status 1

    Re-run with --verbose to see debug info. 

What should I do for the plugin?

My data is “demultiplexed paired-end data”. I did not run the “qiime demux…”, how can I run the demux summarize to view the Demultiplexed sequence counts summary.
I tried the command: qiime demux summarize \ --i-data demux-paired-end.qza \ --o-visualization demux.qzvWhen I go to QIIME 2 view, I only have two-sample sequence counts. How can I do with this correctly?

The quality plots run successfully. I conducted the denoise-paired with the following commands:
"qiime dada2 denoise-paired \ --i-demultiplexed-seqs demux-paired-end.qza \ --o-table table.qza \ --o-representative-sequences rep-seqs.qza \ --p-trim-left-f 0 \ --p-trim-left-r 0 \ --p-trunc-len-f 200 \ --p-trunc-len-r 200"
Then I was trying to build the feature-table summarize, can you give any suggestions about how to build the sample-metadata. tsv for this demux-paired-end data? I have created a .tsv file but really have no clue how to create #sampleId to connect this sample-metadata with the sequence data?

Thanks!

Hi @Bing!

Sorry I think your posts may have gotten mixed up in the moderator-queue, but it looks like you have probably already sorted out the the plot-qualities issues. Please let us know if that isn’t the case.

I think I understand your question, but please let me know if I haven’t.

Now that you have a the results of denoise-paired, you don’t need to explicitly connect the sample-metadata with the sequences (that was already done by demux with your barcodes). Instead what you have is called feature table (produced by denoise-paired) which, in this particular case, has the occurrences of each denoised sequence variant (a.k.a. 100% OTU) for each sample (it is basically a matrix).

What this means is your data is now in the same form as single-end data, so you don’t need to do anything special with your metadata, as your metadata reflects things about your sample, and the feature table ties it back to your representative sequences. It is probably worth pointing out that denoise-paired joins the forward and reverse reads as part of its operation which is why it looks the same as single-end data.

HI @ebolyen
Thank you for the immediate response to my questions and this is so helpful. I did finished all the process following the Moving Pictures Tutorials. I am still kinda confused by the sample metadata.tsv I created. If I built my metadata without any name of the variable in the metadata connected or similar to the table.qza, how the final results will be analyzed? Please help me explain this because I want to make sure I understand it correctly.

My another question is that I have got the demultiplexted paired-end data from our genomic core, how can I run the command to generate a summary of the demultiplexing results? I saw the command in the tutorial is qiime demux summarize \ --i-data demux.qza \ --o-visualization demux.qzv
Is there any way I can run this for the demux paired-end fastq data or I have to request it from the genomic core?

The sample metadata has IDs which must match the IDs in table.qza and a couple other artifacts. If they did not match, we would have no way to relate your actual metadata (in the tutorial things like BodySite etc.) to your computed data such as what sequences were observed and in what abundances.

The table.qza is special in that it has both the IDs from your metadata and the IDs from your observed sequences (representative sequences/denoised sequence variants) so then other methods in the tutorial can use the table to understand the relationships between your samples/metadata and everything else.[quote=“Bing, post:6, topic:350”]
My another question is that I have got the demultiplexted paired-end data from our genomic core, how can I run the command to generate a summary of the demultiplexing results? I saw the command in the tutorial is qiime demux summarize \ --i-data demux.qza \ --o-visualization demux.qzvIs there any way I can run this for the demux paired-end fastq data or I have to request it from the genomic core?
[/quote]

That should be possible, you will just need to import your data as SampleData[SequencesWithQuality], I would check out the importing tutorial and if you have any further questions on that, please feel free to create a new topic on the forum!

Hopefully that answers your questions!

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