Interactive Quality Plot : Danger: Some of the PHRED quality values are out of range

[yinghe]

Hello,

I'm using QIIME2 to analyze my human gut microbiome 16s rRNA sequence data.
After I imported my single-end demultiplexed fastq files and generated a summary of the demultiplexing results, the next step is Deblur, which need to trim the seq first. So I view the demux.qzv and try to find a reasonable trim length. But the plot is really weird and there is a Danger warning at the top of the web view.
Not exactly sure what this error means, or how to get started figuring this out.
Any pointers would be greatly appreciated.

Thanks,
Ying

[thermokarst]

Hi @yinghe, thanks for writing to us. Unfortunately, there is no reliable way for us to know if your sequences are PHRED 33 or PHRED 64 offset, so the default in QIIME is to assume that sequences with quality are PHRED 33. The warning you are seeing seems to indicate that your sequences are actually encoded with PHRED 64 offset quality scores ("Some of the PHRED quality values are out of range. This is likely because an incorrect PHRED offset was chosen on import of your raw data", from the error message in your screenshot). Please see the Importing Tutorial for more discussion about importing your data using the SingleEndFastqManifestPhred64 format, which will convert all of your data to PHRED 33 offset during import. Please note, this process can be slow, but it only needs to happen the one time (during import). Hope that helps, and feel free to follow up with any more questions!

PS - If you have any thoughts about how to reword the Danger message in the summarize visualization, we would be really interested to hear them. Thanks!

[yinghe]

Thank you, Matt.
So, does I have to use the [PairedEndSequencesWithQuality] type for PHRED 64? I used the ‘input-path casava-18-single-end-demultiplexed’ before.

[thermokarst]

You will need to define a manifest file that lists out all of your demultiplexed sequence files:

sample-id,absolute-filepath,direction
sample-1,$PWD/some/filepath/sample1_R1.fastq.gz,forward
sample-2,$PWD/some/filepath/sample2_R1.fastq.gz,forward
...

and save that to a new file (for this example, I will name it se-64-manifest).

Then, you can import the sequences by running something like:

qiime tools import \
  --type SampleData[SequencesWithQuality] \
  --input-path se-64-manifest \
  --output-path demux.qza \
  --source-format SingleEndFastqManifestPhred64

One shortcut to make it easier to do this is to take the QZA that has your first attempt at importing, and export the data from it to get a starter template for the manifest. In your exported directory would be a file called MANIFEST (please ignore all the other files that are exported with it), which you could then copy and use as your new manifest file. You would need to edit the second column and rename it to absolute-filepath, and then edit the entries in that column to become absolute filepaths. Then, you could use this manifest in the import command I provided above.

Hope that helps and good luck! Feel free to write back if you get stuck on anything.

[thermokarst]

Just a quick note, this post originally had a typo in it related to the semantic type specified for import — @yinghe contacted us via direct message, to let us know about this, and I have updated the post to reflect that change. Thanks @yinghe!