Hello, I am new to Qiime2. I have questions about importing sequence data. I have barcode sequences. How can I prepare or generate a barcodes.fastq.gz file? I know convert_fastaqual_fastq.py in Qiime 1 can convert fasta to fastq, but I do not have a QUAL file for the barcodes. Before I import dat…

Hi Tom, Your library prep method differs from the EMP protocol (in which the MiSeq produces separate sequence and barcode fastqs), hence the EMP data importing and demux commands will not work. However, I do believe we can get your data into QIIME2 if you follow the steps below. It sounds like you…

[image] eDNA: I have barcode sequences. How can I prepare or generate a barcodes.fastq.gz file? I know convert_fastaqual_fastq.py in Qiime 1 can convert fasta to fastq, but I do not have a QUAL file for the barcodes. Is your data already demultiplexed? If it's already demultiplexed you can i…

Thanks for your reply. My data is still multiplexed. I know the barcode sequences for each sample, but I do not have a barcodes.fastq.gz file. Can you advise how to get a barcodes.fastq.gz file for my barcodes?

What file format are your barcodes in? Can you post an example of what those look like? Solely knowing what barcode is used for each sample isn't sufficient to demultiplex your data in QIIME 2. You'll need to have a .fastq file containing your sequences, and another .fastq file that has each sequen…

Hi Jai, I only know what the barcode sequences are (e.g. barcode 1: acagca). I think I should give more details about how we generated the data. We have 20 tagged primer pairs (each primer sequence contains a 6-base tag (barcode)). We pooled PCR product of 20 samples (each sample was PCR amplified…

[image] Nicholas_Bokulich: unless if q2_demux.emp_single does not support 6nt barcodes but I don't think this is enforced qiime demux emp-single and qiime demux emp-paired don't have restrictions on barcode length so 6nt barcodes should be fine.

Just for reference, we have an open issue to add support for importing this type of multiplexed sequence data, where the barcodes are contained within the sequences. We'll follow up here when it's available in a QIIME 2 release!

QIIME 2 2017.12 has a new cutadapt plugin which provides demux-single and demux-paired for demultiplexing reads where the barcodes are included within your reads!

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How to prepare the barcodes.fastq.gz file

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