Import multiplexed R1.fastq and R2.fastq with mixed forward and reverse reads + truncate reverse primer

[ebolyen (Evan Bolyen)]

Hi @Martin!

@Martin

The sequencing company suggests (and they do this also in their own data analysis pipeline) to join the reads with qiime1 (join_paired_ends.py) and then to re-orientate all reads in forward direction and remove barcodes (extract_barcodes.py). In the resulting fastq file, the sequences are multiplexed, the forward primer and reverse primer are still present, and the barcodes were extracted into an additional fastq file. From here on, I can import the re-orientated and joined reads in qiime2 using the EMPSingleEndSequences protocol

That is quite an involved workflow! We're still working on a couple of upstream steps (e.g. extract_barcodes.py) so a lot of this is kind of hypothetical:

@Martin

Is it somehow possible to import these forward/reverse mixed-up R1.fastq R2.fastq files using qiime2? (so I could use DADA2 or q2-vsearch for joining of the reads without the need of qiime1)

There is a format for paired-end multiplexed data, however it does presume that your barcodes are in a separate file, which isn't the case without something like extract_barcodes.py.

@Martin

Is it possible to detect the reverse primer, trim it of and delete all the sequences that do not have a correctly matching reverse primer? Either by using a fastq file with joined reads, or after importing the fastq into qiime2? For qiime1, there was the truncate_reverse_primer.py plugin, however this works only with fasta and not fastq.

This is actually something we are looking to have implemented soon, this same situation generally exists for ITS data where your reverse primer ends up on your forward read. I don't think we had really expected to filter via the reverse primer, but it's an interesting idea (though not relevant to ITS which is what we've mostly been thinking about). We're thinking about a cut-adapt plugin, to handle both extract_barcodes.py and truncate_reverse_primer.py, so this could probably fit into that somewhere at some point.

@Martin

The same as in (2) would also be nice for the forward primer: With DADA2 I can trim of the first bases that in most cases correspond to the forward primer. However, in some instances, the forward primer is incorrect and I would rather like to delete the whole sequence, instead of trimming it.

That makes sense, perhaps we could have something that could filter sequences based on primers/adapters which could also handle your reverse-primer filtering scenario.

In short, we basically don't have any of those pieces in QIIME 2 yet, but we're working on it! Thanks for letting us know this is something you need!