I’m new using qiime, so still getting my head around how it works.
just a quick question: when sequences are imported whit a fastq manifest is the output a demultiplexed sequences file?. Example: when importing with either SingleEndFastqManifestPhred33 or PairedEndFastqManifestPhred64 the output is a qza file of either single-end or paired-end depending on the sequences type ( single-end-demux.qza or demux-paired-end.qza). Are these outputs already demultiplexed?
In other words does the importing process includes a demultiplexing step?
Thank you in advance.
Welcome! You are asking all the right questions.
Let’s start at the beginning, when the samples go on the MiSeq. (You might know this already, so feel free to skip ahead.)
- A miseq run costs about $3000, but the cost per sample is much lower because you can put +300 samples on a single run. The processing of barcoding your samples then mixing them together on a single run is ‘multiplexing’.
- The miseq run gives you three important files:
R1, R2, I(forward read, reverse read, and index read that has your barcodes). So you can join up your paired R1 and R2 reads, but most importantly, you will want to separate out your reads based on their barcode, so you can see your +300 samples independently. Any process that un-mixes your reads so you can look at your samples separately is ‘demultiplexing’.
Now back to your questions:
Yes, your collection of fastq files listed in the manifest are already demultiplexed, because they are already separated by sample name.
In this case, when using a manifest file, the samples are already demultiplex, so… technically no. But for other inputs like
qiime tools import --type EMPPairedEndSequences, the samples are not demultiplexed, so then yes, the import step will demultiplex them.
qiime tools import takes many different inputs, but it will always output a qiime artifact (containing demultiplexed reads).
Let me know if that answered your question, or if you have followup questions.
Welcome to Qiime 2!
Thank you very much for the answer, it does make sense now, my sequences are already demultiplexed and I can go ahead with next steps.
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