Importing mixed datasets paired and unpaired FASTQs already cleaned up/QCd

[Nicholas_Bokulich]

I am also not following your question. This experimental setup is not clear to me but here goes:

@mmelendrez

Do I just import them separately

Yes, you will want to process these separately as @thermokarst advised.

@mmelendrez

And if I do that am I able to combine them as one artifact so all 4 can ultimately be compared or leave them as two?

Ultimately you will want to merge these (as @thermokarst indicated) if you wish to compare these samples. However, importing/merging is the least of your worries. If these samples are processed differently, e.g., with different final read lengths (after joining paired-end reads, that is), then each sample will have 100% unique features and you cannot compare using sequence variants. You will need to:

1. use q2-fragment-insertion to compare samples with discontiguous features.
2. assign taxonomy and use taxonomic assignments as features for comparing samples (e.g., alpha, beta diversity, ancom). The issue with this approach is that sequences of different lengths may well be assigned to different taxa, leading to the same problem of unique features...
3. trim your joined paired-end reads to the same length as the single-end reads. In which case you may as well:
4. process all samples as single-end reads with the same parameters.

I personally prefer #4. Trying to compare samples that have been processed with different pipelines/parameters can be a major challenge (and this is not a problem specific to QIIME2).

@mmelendrez

‘alas’ my data is also too large to process with dada2

Your dataset is not too large, and dada2 is in no way the constraint here (unless if I misunderstood). The constraint would be that your computer does not have enough memory.

@mmelendrez

were not equal in terms of FASTQ sequences

What does this mean? Shorter than others?

@mmelendrez

I don’t now how that happens with FASTQ Illumina output but I suspect their prefiltering QC did it.

This is not a default of Illumina, as far as I know, but it sounds like perhaps your sequencing center or service is performing some kind of pre-trimming as part of their QC. I would recommend discussing this with them — you should get the rawest form of the data possible and process entirely within QIIME2. In general, pre-processing with other programs only increases the likelihood that some incompatibilities with QIIME2 can be introduced (e.g., if these programs alter the expected formats or trim sequences enthusiastically).

Good luck!