import multiplexed miseq single-end, barcodes in mapping file

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1

Version: qiime2-amplicon-2024.10

I have an older Illumina miSeq single-end dataset with NO barcodes.fastq.gz file. The barcodes are in the mapping file (see below). I searched the forums and I found the cutadapt code that seems to be what I need demux-single: Demultiplex single-end sequence data with barcodes in-sequence. — QIIME 2 2024.10.1 documentation

But it requires a seqs artifact and that is where I am stuck. I'm not sure how to make a seqs.qza with no barcodes.fastq.qz file. I couldn't find how to make a barcode.fastq.qz file in the forums or tutorials, and I couldn't find out how to make this artifact: --i-seqs ARTIFACT MultiplexedSingleEndBarcodeInSequence

This is the cutadapt code:
qiime cutadapt demux-single \
--i-seqs seqs.qza \
--m-barcodes-file md.tsv \
--m-barcodes-column barcode-sequence \
--o-per-sample-sequences per-sample-sequences.qza \
--o-untrimmed-sequences untrimmed-sequences.qza

This is a portion of my mapping file which is properly formatted:

image
image670×256 12.3 KB

And this is the error trying to import using the EMPSingleEndSequences (which requires a barcode file of course):slight_smile:

qiime tools import --type 'EMPSingleEndSequences' --input-path emp-single-end-sequences --output-path seqs.qza
There was a problem importing emp-single-end-sequences:

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

Any help is appreciated. -Scott

2

Hi Scott,

I think you are half way there!

Here's how to import the data while it's still multiplexed:
(This makes the MultiplexedSingleEndBarcodeInSequence .qza file you need)

qiime tools import \
  --type MultiplexedSingleEndBarcodeInSequence \
  --input-path muxed-se-barcode-in-seq/sequences.fastq.gz \
  --output-path multiplexed-seqs.qza

This method claims to use " 1. one metadata file with a column of per-sample barcodes for use in FASTQ demultiplexing"
but that file is not passed as input! Why is that?

Because it's used by cutadpt in the next step!

qiime cutadapt demux-single \
--i-seqs multiplexed-seqs.qza \ # the file we just imported
--m-barcodes-file md.tsv \
--m-barcodes-column barcode-sequence \
--o-per-sample-sequences per-sample-sequences.qza \
--o-untrimmed-sequences untrimmed-sequences.qza

Let me know if that works!