Low read abundance after demultiplexing

Hi! new Qiime2 user here!
I've been dealing with an issue in the demultiplexing step. I'm trying to repeat an analysis of a previously published paper. I've got their forward and reverse fastq files, barcodes, and metadata. after demuxing and getting the summary table, the read count for each sample is extremely low(max 43000, min 73 !).
I've checked different flags( also –p-golay-error-correction-details ), and the script pasted below is the only way that I even get an output :

qiime demux emp-paired
--i-seqs emp-paired-end-sequences.qza
--m-barcodes-file barcodes.tsv
--m-barcodes-column BarcodeSequence
--o-per-sample-sequences demux.qza
--o-error-correction-details demux-details.qza

qiime demux summarize
--i-data demux.qza --o-visualization demux.qzv

any idea why this might be?
per-sample-fastq-counts.tsv (1.7 KB)

Hello @Anahid_Amani_Moghada,

What is the output when you run gunzip -c <file> | wc -l on either the forward or reverse reads file? This (divided by four) will tell you how many demultiplexed reads you would expect in the best case.

Can you also share the demux-details.qza file?

Hi @colinvwood
the command gives "62479544" back to me. also in the paper they state that they get > median depth of sequencing of 12,385 reads per sample (IQR = 10840-16136). there are 100 samples
the details file was too large to upload here but here is the link to google drive:

thanks for your help!

Hello @Anahid_Amani_Moghada,

Providing both the --p-rev-comp-barcodes flag and the --p-rev-comp-mapping-barcodes flag at the same time should have the same effect as providing neither. Have you tried just one or the other?


@colinvwood I've checked one of them before but not the other one, it worked, thanks a million!

1 Like

Hello @Anahid_Amani_Moghada,

Yes, please post that separately so that in the future people looking for help on this topic won't be distracted by a different one.