Which import parameters should I use

Hello, I am confused about the first step about imported data. I sequenced both 16SrRNA and fungal ITS. Both of them use EMP standard primers. I have three fastq files. F, R, and barcode fastq files stored in /raw/ directory.

For 16S rRNA, I use the command like this

Imported 1st Bacterial 16S sequencing data

qiime tools import --type EMPPairedEndSequences --input-path raw --output-path $PWD/1st.bacteria.sequences.qza

#Imported 2nd Bacterial 16S sequencing data

qiime tools import --type EMPPairedEndSequences --input-path raw --output-path $PWD/2nd.bacteria.sequences.qza

Then, use qiime demux emp-paired to do the next. It works very well.

However, for fungal data, I couldn’t even import it using EMPPairedEndSequences. You know the EMP fungal barcode is one the Reverse sequences, but I have even get that step. I can’t even import the data.

Can any one tell me?
1. “EMPPairedEndSequences” Does this only work for EMP bacteria? Why it doesn’t work for fungal EMP data?

2. What parameter should I use? “FeatureData[PairedEndSequence]” or "MultiplexedPairedEndBarcodeInSequence"

Hey @moonlight!

EMP-formatted data will always have the barcode-reads extracted as a third file (barcodes.fastq.gz), so the read direction it was sequenced on shouldn’t matter. Can you provide a bit more detail about the format of these data, it sounds like they might not actually be EMP-formatted reads.

Hi, For my fungal data. I have unzip the .gz file? I have to use gz format, right? or fastq is alright too.
Also, does it matter the barcode on the forward or reverse sequences

Hi @moonlight - please provide a bit more detail about these data - in particular provide some info about the files you have, and what they represent. Maybe a screenshot or two?

It depends on what is zipped, but in general this shouldn’t be necessary.

It depends on the import format used. For EMP-format sequences, you will need to import fastq.gz files.

Not for EMP-formatted sequences, because the barcodes will be in their own file, not on the forward or reverse reads files.

Keep us posted!

I highly recommend you spend some time reviewing this tutorial, it will give you a good overview of importing in QIIME 2:

https://docs.qiime2.org/2020.2/tutorials/importing/