Errors in importing fastq files

Technical Support
1

Hello,

I am having some trouble importing some fastq files into qiime. Specifically, I have two fastq files, an R1 and an R2 from a toy dataset. Technically, they are not multiplexed samples, but they contain amplicons from multiple variable regions of the 16S, and I would like to use cutadapt downstream to split the sample into individual variable regions via the primer sequence. I successfully imported the files into qiime as SampleData[PairedEndSequencesWithQuality] using the below command:

qiime tools import
–type ‘SampleData[PairedEndSequencesWithQuality]’
–input-path manifest.txt
–output-path demux.qza
–input-format PairedEndFastqManifestPhred33V2

My manifest is pretty simple (all tab-separated values):

sample-id forward-absolute-filepath reverse-absolute-filepath
toy /Users/garlic/Documents/MULTI_REGION/Example_L001_R1_001.fastq /Users/garlic/Documents/MULTI_REGION/Example_L001_R2_001.fastq

But when I tried to split the files by primer into different variable regions, I received this error:

(1/1) Invalid value for “–i-seqs”: Expected an artifact of at least type
MultiplexedPairedEndBarcodeInSequence. An artifact of type
SampleData[PairedEndSequencesWithQuality] was provided.

So, I then tried to import the sequences as MultiplexedPairedEndBarcodeInSequence:

qiime tools import
–type MultiplexedPairedEndBarcodeInSequence
–input-path manifest.txt
–output-path demux.qza
–input-format PairedEndFastqManifestPhred33V2

But I received an error:

No transformation from <class ‘q2_types.per_sample_sequences._format.PairedEndFastqManifestPhred33V2’> to <class ‘q2_types.multiplexed_sequences._format.MultiplexedPairedEndBarcodeInSequenceDirFmt’>

Can someone please advise me on what I am doing wrong? Is it possible to import these as MultiplexedPairedEndBarcodeInSequence?

Thanks!

2

Hello @garlic, that error message is saying artifacts of type MultiplexedPairedEndBarcodeInSequence want to be in the MultiplexedPairedEndBarcodeInSequenceDirFmt format, but you are attempting to import your artifact explicitly in the PairedEndFastqManifestPhred33V2 format, which Qiime2 does not know how to turn into the format it wants.

That in mind, have you tried running that second import command without passing the --input-format explicitly? This should allow Qiime2 to import the artifact in the format it wants the artifact to be in. The command would look something like this

qiime tools import
–type MultiplexedPairedEndBarcodeInSequence
–input-path manifest.txt
–output-path demux.qza

just the same as what you ran minus the --input-format.