Hi all,
I recently obtained 16S sequence data from ~280 samples for my dissertation.
I got the data from the sequencer in both demultiplexed and non-demultiplexed form.
I'm most familiar with data that is already demultiplexed, but my collaborator said I should start with non-demultiplexed data because QIIME2's demultiplexing is better than Illumina's.
I've taken the demultiplexed data through DADA2 and into some alpha diversity analysis with no problems. Of the 280 samples, only a handful had less than 100 reads, and only 1 completely dropped out after DADA2. From what I can tell, the dataset as a whole is sound and should be good enough to proceed with.
However, when I have tried to import and demultiplex the other (non-demultiplexed) version of the data, I only end up with 24 samples. This version of the data consists of two large files containing all of the forward and reverse reads, respectively. I also have a file with all of the barcodes that I produced from the information given to me by the sequencing center.
Below is the code I have used to get this far, and I have also attached my demux.qzv. Thank you for any insight or ideas!
demux.qzv (300.8 KB)
qiime tools import
--type MultiplexedPairedEndBarcodeInSequence
--input-path fastq
--output-path multiplexed-seqs.qza
qiime cutadapt demux-paired
--i-seqs multiplexed-seqs.qza
--m-forward-barcodes-file barcodes.txt
--m-forward-barcodes-column forward
--m-reverse-barcodes-file barcodes.txt
--m-reverse-barcodes-column reverse
--o-per-sample-sequences demux.qza
--o-untrimmed-sequences untrimmed.qza