Import joined data

User Support
1

Hello,
I can not import my raw sequence in QIIME2 because I need to use FLASH to combine the pairs of read files with --allow-outie option after trimm and quality filter.

-O, --allow-outies      Also try combining read pairs in the "outie"
                      orientation, e.g.

                           Read 1: <-----------
                           Read 2:       ------------>

                      as opposed to only the "innie" orientation, e.g.

                           Read 1:       <------------
                           Read 2: ----------->

                      FLASH uses the same parameters when trying each
                      orientation.  If a read pair can be combined in
                      both "innie" and "outie" orientations, the
                      better-fitting one will be chosen using the same
                      scoring algorithm that FLASH normally uses.

Then, I can import my mergeALLreads.fasta into rep-seqs.qza with qiime tools import (type 'FeatureData[Sequence]').

But I don't know how is the next step, I mean, I have representative sequences (rep-seqs.qza) but I don't have the table.qza. Since qiime2 is working, OTUs has changed to ASVs, so, If I want to use dada2 or deblur for denoising, instead of OTU picking methods, I will need to start with the raw fastq, but if I can't do it?

Is there any procedure that the qiime2 starting point is joined-reads (rep-seqs.qza)?

Thanks for the help!

2

Have your sequences undergone ANY quality control other than joining?

I believe the recommend way is to use the manifest format of importing reads.

If you import it as single end reads with either phred33 or 64 (whichever it is, if you're unsure just try both and use whichever one works).

You then should be able to use whatever you want to create tables and representative sequences.

3

I know that how to work with qiime and vsearch, but unfortunately, vsearch hasn't any option to join OUTIE orientation, because of this I need to use FLASH, and I want to import my data in another point of pipeline if it's possible.
In any case thank you.

4

Hi Cristina,
after the import of the merged sequences, you still need to perform the denoising step, then you will have the rep-seqs.qza and table.qza. Given you using pre-merged sequences, the suggested way is to use deblur for the denoising step. You may want to perform a quality control step as suggested by Jono before the denoising step.

Good luck,
Luca

2 Likes
5

This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.