How to demultiplex paired-end illumine reads with in-line barcodes?

I have in-line barcoded Paired-end illumine ITS reads. Basically, there are 6bp barcode sequences at the immediate upstream 5’end of ITS3 and ITS4 primer respectively. First, I used the from Qiime1 ( that can chop off the first 6bp from both forward and reverse reads, then merge together to 12bp and spit out barcodes.fastq, read1.fastq and read2.fastq. After I inspected all three files, I converted them to barcodes.fastq.gz, forward.fastq.gz and reverse.fastq.gz to import them to Qiime2 by using “qiime tools import --type EMPPairedEndSequences”. My last step is to demultiplex using “qiime demux emp-paired --m-barcodes-category BarcodeSequence”. There is no error message, but only <100 reads parsed out. I checked my barcodes-file, it looks like all right. Is there any option to be turned out to see intermediate step of demux? Is there a better to demultiplex inline barcoded PE reads? Many thanks.

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Hi @hotblast,

Take a look at cutadapt demux-paired. Based on your description, it sounds like this might be the plugin for you (unless if you are using dual-indexing, in which case see below) and will sidestep the need to use qiime1.

However, that probably won’t fix the error that you are running into… here are my thoughts:

Based on your description, everything sounds correct so your low demux yields make me think of one detail in particular:

How do these barcodes in the barcode file compare to the mapping file? You may want to try adding the --p-rev-comp-barcodes parameter to see if that improves things.

It also sounds like you may be using a dual-indexing scheme, in which case this thread might also be useful to you.

Thanks for your suggestion. The problem have been solved after i applied “–p-rev-comp-mapping-barcodes”. The reason is due to our in-house inline dual index barcode used.
Thanks again for your quick response, you guys are great!!!


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