Help Importing demultiplex paired end files with barcodes in head (only two files, files not separated by sample)

Hi All,

I am requesting so assistance with importing fastq files I recently received. There are only two files that contain the Forward and Reverse reads of all of my samples. I believe that the sequences have already been demultiplexed. Also these sequences have the barcode included in the header, and I do not have a separate barcodes.fastq file.

I am struggling with how to import these data as none of the tutorials on the Import help page seem to apply.

Here is some of the code I have run:

qiime tools import --type EMPPairedEndSequences --input-path 16S --output-path emp-paired-end-sequences.qza

There was a problem importing 16S:

Missing one or more files for EMPPairedEndDirFmt: 'barcodes.fastq.gz’


qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path 16s --output-path demux-paired-end.qza

There was a problem importing 16s:

Missing one or more files for SingleLanePerSamplePairedEndFastqDirFmt: '.+_.+_L[0-9][0-9][0-9]_R[12]_001\.fastq\.gz

Thank you for your help!


Hey there @Jane_Lucas!


Maybe not - if there are only two files with all the samples in them, that indicates that things are still multiplexed.

Not having a separate barcodes file is fine, we have plenty of options to get around that, but they require that the barcodes are still present in the read itself, rather than in the header. Is it possible the barcode is in both the header and the read? If so, then check out this community tutorial for demultiplexing using q2-cutadapt.

If the barcodes are only in the header, then you will need to find a way to demultiplex these reads prior to importing into QIIME 2, as we have no way to demux reads based on headers. Once demuxed, you could import using the fastq manifest.

Keep us posted! :qiime2: :t_rex:

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