Demultiplexing EMP-Paired-sequences Error: Sequence counts dropped dramatically

I encountered the same problem as the user GillSie Sierra G ( Demux EMPPaired Error without rev-comp-mapping-barcode - User Support - QIIME 2 Forum), and I read and implemented his solution, but it didn't work for me.
So, I have to create a new post to ask for help.

For studying Qiime2, I have forward.fastq.gz, reverse.fastq.gz, barcodes.fastq.gz and mapping_file.tsv downloaded from Qiita (ID 11635). I think it matches the type EMP-paired and it looks like that there are no barocdes and primers in forward.fastq.gz and reverse.fastq.gz.

Here are my operation steps and error reporting
Step1: I have imported my files as EMP-paired using the command:
qiime tools import
--type EMPPairedEndSequences
--input-path /result/emp-paired-end-sequences
--output-path /result
I succeed.
Step2: I used the following code to demultiplex and got the "demux03.qza" successfully.
qiime demux emp-paired
--i-seqs emp-paired-end-sequences.qza
--m-barcodes-file mapping_file.txt
--m-barcodes-column barcode
--o-per-sample-sequences demux03.qza
--o-error-correction-details demux-details03.qza
qiime demux summarize
--i-data demux03.qza
--o-visualization demux03.qzv
However, when viewing "demux-full.qzv", the sequence counts dropped dramatically after the first two samples and looked so strange.

I have try the code "--p-no-golay-error-correction" and "--p-rev-comp-barcodes" to replace and I failed (I put my attempts and errors below).



What did I do wrong? What should I do?
Any suggestion will help me :pray: :pray: :pray:, and I will be very grateful !!
Sincere greetings!!

Good afternoon @GRuii,

Welcome to the forums! :qiime2:

I think Qiita provides counts per sample, so you could check that to see if the sample counts are truly wrong, or if some samples simply have more reads inside of them.

You could also extract and summarize barcode counts inside the barcodes.fastq.gz file to see how they compare, but that's a lot more manual processing.

Thanks for your reply,
I have checked the counts provided by Qiita, and they are truly more than that in our outcomes.
Also, I compared the number of lines contained in barcodes.fastq.gz, forward.fastq.gz and reverse.fastq.gz. The three raw files have the same number of lines.
I am not sure if I get what you mean. Could you tell me what should I do next?
Anxious anticipates your reply.

Hi @GRuii,

Qiita reports that both rev_comp_barcode and rev_comp_mapping_barcodes was specified. What happens if you rerun using --p-rev-comp-barcodes and --p-rev-comp-mapping-barcodes?


Screen Shot 2023-03-06 at 8.59.26 AM

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Thank you very much for your time and explanation.
The problem has been well solved when I rerun using --p-rev-comp-barcodes and --p-rev-comp-mapping-barcodes.
However, I still have a confusion. Could you please tell me how can I know whether there have rev_comp_barcode and rev_comp_mapping_barcodes as shown in your reply.
Sincere greetings.

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If you select the split libraries node in Qiita, it will show you the parameters applied. Does that make sense?


I found it! Thank you very much !!!!! :smiling_face_with_three_hearts: :smiling_face_with_three_hearts: