how to import these reads: Mixed-oriented or not?

[Wieneke]

@colinbrislawn

As far as I can tell, these are normal paired end Illumina sequencing reads.
Files ending in _1.fastq.bz2 are your forward reads (confirmed by 1:N: in the read header), and files ending in _2.fastq.bz2 are your reverse reads (confirmed by 2:N: in the header).

But how can it be then that there are reverse primers in the 1:0 reads and forward primers in the 2:0 reads?

I used the "standard" pipeline with DADA2 (version 2021.8) renaming the sequences to forward and reverse in directory muxed-pe-barcode-in-seq-L26
qiime tools import --type MultiplexedPairedEndBarcodeInSequence --input-path muxed-pe-barcode-in-seq-L26 --output-path multiplexed-seqsL26.qza
then demultiplexing
demultiplexed-seqsL26.qzv (317.4 KB)

qiime cutadapt demux-paired --i-seqs multiplexed-seqsL26.qza --m-forward-barcodes-file metadata_library26.tsv --m-forward-barcodes-column forward-barcode-seq --m-reverse-barcodes-file metadata_library26.tsv --m-reverse-barcodes-column reverse-barcode-seq --o-per-sample-sequences demultiplexed-seqsL26.qza --o-untrimmed-sequences untrimmedL26.qza --verbose
with barcode file

denoising-statsL26.qzv (1.2 MB)