Import of reverse reads

[Mandy]

hello,

Since I’ve used DADA2 denoise single for my 16S data I have been instructed to evaluate my forward and reverse reads seperately.
So I’ve been trying to import only my reverse reads.
I made a new directory, changed my manifest by only keeping the reverse path-ways, and used the following command:
qiime tools import
–type EMPSingleEndSequences
–input-path MANIFEST_Mondsee_16S_rev.txt
–output-path p01_reads.qza

that reulted in the following error: There was a problem importing MANIFEST_Mondsee_16S_rev.txt:

Importing ‘EMPSingleEndDirFmt’ requires a directory, not MANIFEST_Mondsee_16S_rev.txt

• So I used the same command but with my directory as an input path, which results in this error: Invalid value for “–input-path”: Path “DADA2_denoise_single_rev” does
  not exist.

• (I checked for spelling mistakes, there are none)

• then I tried to use the following command, which reults in different errors every time I use it:
  qiime tools import
  –type ‘SampleData[SequencesWithQuality]’
  –input-path …/16S/MANIFEST_Mondsee_16S_single_rev.txt
  –input-format CasavaOneEightSingleLanePerSampleDirFmt
  –output-path demux-single-end.qza

-My reads are not joined, and they are already demultiplexed, does anyone have an idea how I could move forward?
Am I looking in the wrong direction regarding the commands I use?
-the only thing I could think off is switching my forward and reverse file paths in my Manifest file to “make it use” only the first set of reads?

Thank you for any help, as always!!
cheers.

[SoilRotifer (Mike Robeson)]

Hi @Mandy,

Seems like you are on the right track given the following trick:

Miseq paired-end workflow

Hi @wym199633, I suspect that you should be able to trick QIIME 2 to import the reverse reads as forward reads by using the manifest format approach. That is, just set the direction column for these reads to forward for all your R2 reads, and do not include your R1 reads. I can not speak to any sequence data validation steps that occur via this or other import approaches. Perhaps @ebolyen, @thermokarst, or someone else can provide some insight on weather this will work, or if there is a more a…

But not the warning about naming:

Miseq paired-end workflow

Hi @wym199633, @SoilRotifer is correct — you can trick QIIME2 into using the reverse reads as forward reads by using the manifest file format. With the exception of the CASAVA1.8 paired-end format, which expects specific filename patterns to differentiate the forward and reverse reads, all other formats in QIIME2 are direction-agnostic.

-Mike