Create demux.qza from provided merged and trimmed paired-end files

[microbiomeAnalyst]

Hello. I have received fastq files which are demultiplexed and have already been trimmed and merged (originally paired-end). Is there a qiime2 command which I can use to create the demux.qza? I tried the cassava single lane command but I’m presuming it expects only a forward sequence.

Please let me know if I can add any clarity to my question.

Thank you.

[colinbrislawn (Colin J Brislawn)]

Hello!

One option would be to import this data as if it was forward reads, by using the fastq-manifest-format.

Another option would be to get the original raw files, and perform the pairing and quality directly inside of Qiime.

Colin

[ebolyen (Evan Bolyen)]

Just to add on to that, set the --type to SampleData[JoinedSequencesWithQuality], it can be used with any of the single-end formats (casava or manifest-style) since the representation is the same.

[microbiomeAnalyst]

Thank you for your reply. Using the following qiime casava command…

qiime tools import
–type ‘SampleData[JoinedSequencesWithQuality]’
–input-path /media/sequences
–source-format CasavaOneEightSingleLanePerSampleDirFmt
–output-path /media/sequences/demux-joined.qza

I received the following error…
There was a problem importing /media/sequences:

Missing one or more files for CasavaOneEightSingleLanePerSampleDirFmt: ‘.+_.+_L[0-9][0-9][0-9]_R[12]_001\.fastq\.gz’

My preference would be to use casava. I’m not certain how to troubleshoot from here, so any direction would be appreciated.

[microbiomeAnalyst]

Hello. I just realized that I may need a manifest file in order to use casava with joined fastq files I researched manifest file formats and could only find the following example format. If this is the correct format, both the forward and reverse sequences map to the the sample-id which seems correct. Where would I place the manifest file designation in the casava format? Thank you!

sample-id,absolute-filepath,direction
AFF3G,/mnt/lustre/hall/mlo1011/qiime_practice/16s_raw_practice/AFF3G_TCCAAAGTGTTC_L001_R1_001.fastq.gz,forward
AFF3G,/mnt/lustre/hall/mlo1011/qiime_practice/16s_raw_practice/AFF3G_TCCAAAGTGTTC_L001_R3_001.fastq.gz,reverse

[Nicholas_Bokulich]

@microbiomeAnalyst

Hello. I just realized that I may need a manifest file in order to use casava with joined fastq files I researched manifest file formats and could only find the following example format

Yes, as @colinbrislawn and @ebolyen advised above, you should use a manifest file. I am re-sharing the link that Colin originally posted:
https://docs.qiime2.org/2018.4/tutorials/importing/#fastq-manifest-formats

@microbiomeAnalyst

Where would I place the manifest file designation in the casava format?

It looks like you have written out an example of a manifest file for paired-end reads. Save to a text file, let's call it pe-64-manifest. Then import with this command:

qiime tools import \
  --type 'SampleData[PairedEndSequencesWithQuality]' \
  --input-path pe-64-manifest \
  --output-path paired-end-demux.qza \
  --source-format PairedEndFastqManifestPhred64

Note that I have specified as paired-end data, because it looks like you list the paired-end reads in your manifest file. If you do want to use the trimmed/joined reads as you originally declared, you can follow the import instructions given here.

Good luck!