Hi @Rishikesh12,
Basically what this error is saying is that your input for qiime cutadapt demux-paired is already demultiplexed. You need to input multiplexed data into qiime cutadapt demux-paired so that the command can demultiplex your data.
I can see two ways this possibly happened. However I am not sure exactly what steps you took given the information you provided.
- You imported already demultiplexed data. If this happened you would probably not have barcodes in your sequences and you would just skip the
qiime cutadapt demux-pairedcommand. - You imported multiplexed sequences and then you ran a command that demultipexed your sequence. Then you used cutadapt trim-paired and now you are trying to demultiplex again.
If this is the case, I would highly suggest that you re-read the Demultiplexing and Trimming Adapters from Reads with q2-cutadapt tutorial that I linked above and here. These steps need to be followed in a specific order or else the types of inputs are going to be wrong like you see in the error message you sent.
In order to help you more on this, I would need to know all the commands that you ran to get to this error. Please list all of them in your next reply.
Hope this helps!
