Invalid Filepath: Qiime feauture classifier and pre-formated rescript seq file

[fabipc (Fabiola)]

Hello,

I am attempting to use the ReScript preformatted sequence and taxonomy files (here: SILVA 16S) to extract the amplicon region from our reference database using my primer set. However, when I attempt to do so, it keeps giving me the error.

There was a problem with the command:
(1/1) Invalid value for '--i-sequences': 'silva-138-99-seq.qza' is not a
valid filepath

The files are all located in the same folder. I am attempting to run the script, so I am confused about the error. Can you please help?

module load qiime2/2022.2
source activate qiime2-2022.2

#Using pre-formatted SILVA reference sequence and taxonomy files here that were processed using RESCRIPt.
#extract the amplicon region from our reference database. (V4-V5 Region)

qiime feature-classifier extract-reads
--i-sequences silva-138-99-seq.qza \ #Pre formatted full length seq files
--p-f-primer GTGYCAGCMGCCGCGGTAA
--p-r-primer CCGTCAATTCCTTTRAGTTT
--p-n-jobs 2
--p-read-orientation 'forward'
--o-reads silva-138.1-ssu-nr99-seqs-515f-926r.qza

Here is the directory where I am running the above script and the files within the folder

[fabipc (Fabiola)]

So weirdly enough, I decided to rerun the script but change/remove some parts of the script to what my original script used to look like (previous versions of SILVA database, not the script used in the ReScript tutorial), and it worked.

However, if you have any idea as to why it worked w/o those 2 lines, I would really appreciate the knowledge.

This is what I changed; removed read orientation and number of jobs

qiime feature-classifier extract-reads \
--i-sequences silva-138-99-seqs.qza \
--p-f-primer TGYCAGCMGCCGCGGTAA \
--p-r-primer CCGTCAATTCCTTTRAGTTT \
--p-min-length 1 \
--o-reads silva-138.1-ssu-nr99-seqs-515f-926r.qza

[Nicholas_Bokulich]

Hi @fabipc ,
It looks like the spelling is inconsistent between your command that worked and the one that did not:

@fabipc

(1/1) Invalid value for '--i-sequences': 'silva-138-99-seq.qza' is not a

@fabipc

--i-sequences silva-138-99-seqs.qza \

So it looks like you just had a typo in the first instance, but fixed it in the second.

I hope that clarifies!

[fabipc (Fabiola)]

Omg! That was a dumb mistake. Thank you for that