I have a fastq sequence file (have both multiplexed and demultiplexed files) from ion torrent.In total there are 192 samples in the sequence with 2 barcodes (16s and ITS). 96 samples with 16s primer and 96 samples with ITS primers and both the 16s and ITS primers are attached to the same barcodes. I find it difficult in Qiime 2 to import or analyse the data. In Qiime1 I can just change the primer sequence in mapping file and either 16s or ITS primers get excluded. Is there a simiar method to do this in Qiime2 as well?
Is it possible to separate the sequence file based on primers and do the downstream analysis. I have been trying different methods but couldn’t able to figure out.
Kindly advise how to proceed with my sequence data.
This should not be an issue to import, and should cause minor issues with processing.
Since 16S and ITS are quite dissimilar, you can use alignment-based filtering to remove these. Two options:
deblur performs positive filtering as part of the pipeline (i.e., it removes sequences that do not roughly resemble the reference database). This will NOT work for you, however, as I believe deblur is not appropriate for Ion Torrent data.
You can use qiime quality-control exclude-seqs to perform this same type of filtering. This would occur after denoising/OTU clustering.
So in your case, use dada2 denoise-pyro to denoise (or use OTU clustering if you prefer), then use exclude-seqs to separate out 16S and ITS sequences.
I did the exclude-seqs step after denoising my sequences. If I am not wrong I should use the rep-seqs.fasta file I got as an output from the denoising step as an input here. If that is so, I understand that I will get the new file from the exclude.seqs with 16s and ITS sequences. But how do I get the OTU table for my 16s and ITS sequences? I understand that there is one OTU table output from the denoise-single.
I used following commands to separate the 16s and ITS sequences
Not to denoise, just to separate the sequences based on primers (as 16s and ITS). I tried running split_libabries.py, but it didn’t work since the command doesn’t recognise the forward primers. As my data is single-end reads I am unable to use the reverse primer truncate_remove option. If I do that, I lose lot of sequences.
if I have to do that in Qiime2 and coming back to my problem, which sequence output file I should use as an output for exclude -seqs command? I only get one output sequence file from denoise-single command. That is rep_seqs.qza file. In the denoise folder I only get some stat file.
I think I finally understand what you are asking. You need to filter your feature table to include only the features that pass exclude-seqs (to get an ITS-only and 16S-only feature table). To do this, use qiime feature-table filter-features, using the correct sequence files as metadata inputs to that command.