I am trying to obtain 16S diversity form my samples. From my raw data, I have trimmed and merged my forward and reverse reads with cut adapt and pandaseq. My outputs are now .fasq files. Could I now start demultiplexing using “qiime demux amp-single” as input has to be in .qza I am not sure how to start from there. I am working with Paired ends, how can I continue from here?
I found the easiest way to do this was to build a manifest, and then import the sequences as as a “SampleData[JoinedSequencesWithQuality]” type. My import command for vsearch joined sequences looks like this:
where I defined my $manifest_path and $art_path as bash variables.
Then, I suggest following the Alternate methods of read joining tutorial from the Viewing a summary of the joined reads step. Please note that you can’t use Dada2 with pre-joined reads, since Dada2 uses read joining as part of their quality filtering, where as Deblur will accept it.
I have created the manifest and run
qiime tools import
–input-path manifest.csv
–output-path ./ready_seqs.qza
–type ‘SampleData[JoinedSequencesWithQuality]’
–source-format PairedEndFastqManifestPhred33
using Paired end instead of single reads as I have both forward and reverse per sample, but Ihave the following error:
An unexpected error has occurred:
No transformation from <class ‘q2_types.per_sample_sequences._format.PairedEndFastqManifestPhred33’> to <class ‘q2_types.per_sample_sequences._format.SingleLanePerSampleSingleEndFastqDirFmt’>
Do you have any idea what does this mean?
thanks
E
You mentioned above that these reads are pre-joined, which means the --source-format should be SingleEndFastqManifestPhred33, as @jwdebelius mentioned (not PairedEndFastqManifestPhred33).
This seems to be at odds with this statement though:
Are you talking about two different datasets here? If they are pre-joined (what you called “merged”), then you wouldn’t have a forward and reverse read per sample. Can you please clarify? Thanks!
Apologies for the confusion, I started working with the joined (already merged samples), but then I have read from other forum posts that in order to use dada, and quality filtering it is advisable to work with the initial forward and reverse reads.
Doing so, I have decided to start with the initial forward and reverse form the different samples, using:
**Forward and reverse reads must be provided exactly one time each for each sample. The following samples had forward but not reverse read fastq files: ECsample1_R1.fastq.gz, ECsample2_R1.fastq.gz + ALL THE SAMPLES
Is there a problem on the manifest? I have checked names and paths and it seems to be ok.
Should I check something else?
Thanks