Large portion of Cyanobacteria

[Sabrin (Abdelghany)]

@SoilRotifer,

Could you please check if my codes for merging the NCBI and SILVA rep.seqs and tax. is correct, step 5&6 so simply merging both corresponding seqs and taxonomy files from both NCBI and SILVA?

qiime rescript get-ncbi-data \
--p-query '33175[BioProject] OR 33317[BioProject]' \
--o-sequences /user/asga9989/NCBI/ncbi-refseqs-unfiltered.qza \
--o-taxonomy /user/asga9989/NCBI/ncbi-refseqs-taxonomy-unfiltered.qza

qiime rescript filter-seqs-length-by-taxon \
--i-sequences /user/asga9989/NCBI/ncbi-refseqs-unfiltered.qza \
--i-taxonomy /user/asga9989/NCBI/ncbi-refseqs-taxonomy-unfiltered.qza \
--p-labels Archaea Bacteria \
--p-min-lens 900 1200 \
--o-filtered-seqs /user/asga9989/NCBI/ncbi-refseqs.qza \
--o-discarded-seqs /user/asga9989/NCBI/ncbi-refseqs-tooshort.qza

qiime rescript filter-taxa \
--i-taxonomy /user/asga9989/NCBI/ncbi-refseqs-taxonomy-unfiltered.qza \
--m-ids-to-keep-file /user/asga9989/NCBI/ncbi-refseqs.qza \
--o-filtered-taxonomy /user/asga9989/NCBI/ncbi-refseqs-taxonomy.qza

qiime rescript evaluate-fit-classifier \
--i-sequences /user/asga9989/NCBI/ncbi-refseqs.qza \
--i-taxonomy /user/asga9989/NCBI/ncbi-refseqs-taxonomy.qza \
--o-classifier /user/asga9989/NCBI/ncbi-refseqs-classifier.qza \
--o-evaluation /user/asga9989/NCBI/ncbi-refseqs-classifier-evaluation.qzv \
--o-observed-taxonomy /user/asga9989/NCBI/ncbi-refseqs-predicted-taxonomy.qza

qiime feature-table merge-seqs \
--i-data /user/asga9989/Taxonomy_output/training-feature-classifiers/ silva-138-ssu-nr99-seqs-derep-uniq.qza \
--i-data /user/asga9989/NCBI/ncbi-refseqs.qza \
--o-merged-data /user/asga9989/NCBI/SILVA-NCBI-merged-rep-seqs.qza

qiime feature-table merge-taxa \
--i-data /user/asga9989/Taxonomy_output/training-feature-classifiers/silva-138-ssu-nr99-tax-derep-uniq.qza \
--i-data /user/asga9989/NCBI/ncbi-refseqs-taxonomy.qza \
--o-merged-data /user/asga9989/NCBI/SILVA-NCBI-merged-rep-taxonomy.qza

qiime feature-classifier fit-classifier-naive-bayes
--i-reference-reads /user/asga9989/NCBI/SILVA-NCBI-merged-rep-seqs.qza
--i-reference-taxonomy /user/asga9989/NCBI/SILVA-NCBI-merged-rep-taxonomy.qza
--o-classifier /user/asga9989/NCBI/SILVA-NCBI-mergerd-classifier.qza

qiime feature-classifier classify-sklearn
--i-classifier /user/asga9989/NCBI/SILVA-NCBI-mergerd-classifier.qza
--i-reads /user/asga9989/atra-rep-seqs.qza
--o-classification /user/asga9989/NCBI/atra-vs-SILVA-NCBI-classifier-taxonomy.qza

qiime metadata tabulate
--m-input-file /user/asga9989/NCBI/atra-vs-SILVA-NCBI-classifier-taxonomy.qza
--o-visualization /user/asga9989/NCBI/atra-vs-SILVA-NCBI-classifier-taxonomy.qzv

[SoilRotifer (Mike Robeson)]

Hi @Sabrin,

Everything looks fine to me. However, know that GenBank and SILVA use slightly different taxonomic nomenclature and also use different prefixes. Prior to merging the taxonomy, you'll likely want to run rescript edit taxonomy ... as I've described here:

DADA2 and Deblur outputs are extremely different

Yes there is. You should be able to follow one of these tutorials: From here you should be able to run qiime feature-table merge-taxa... and qiime feature-table merge-seqs ... to combine the GenBank and SILVA data. Note that, prior to merging, there is a chance you'd have to run qiime rescript edit-taxonomy ... on the fetched NCBI taxonomy data to change the k__ prefixes to d__, so that it matches SILVA. Assuming you are downloading the standard 7 ranks (kpcofgs). Otherwise you'll have to …

-Mike

[Sabrin (Abdelghany)]

@SoilRotifer yes true, I got different prefixes in my final classification.

First question, what if I want to keep the short reads and not filter them, then can i just skip step 2. qiime rescript filter-seqs-length-by-taxon and step 3. and just use ncbi-refseqs-unfiltered.qza and ncbi-refseqs-taxonomy-unfiltered.qza for downstream analysis of step 4.qiime rescript evaluate-fit-classifier ?

second question please, I am trying to use qiime rescript edit-taxonomy now to fix different prefixes issue. So I should chang the prefix K__, in NCBI taxonomy file before merging it to the SILVA taxonomy file like that,

qiime rescript edit-taxonomy
--i-taxonomy ncbi-refseqs-taxonomy-unfiltered.qza
--p-search-strings 'k__Bacteria'
--p-replacement-strings 'd__Bacteria'
--o-edited-taxonomy ncbi-refseqs-edited-taxonomy-unfiltered.qza

Finally, Is there is a simple tutorial I can follow to export my files into R formats for further visualizations please.

Thanks a lot for your continuous help.

[SoilRotifer (Mike Robeson)]

@Sabrin

First question, what if I want to keep the short reads and not filter them, then can i just skip step 2. qiime rescript filter-seqs-length-by-taxon and step 3. and just use ncbi-refseqs-unfiltered.qza and ncbi-refseqs-taxonomy-unfiltered.qza for downstream analysis of step 4.qiime rescript evaluate-fit-classifier ?

You can do whatever you like to suit your needs. The tutorial just simply offers a series of examples to process your data.

@Sabrin

second question please, I am trying to use qiime rescript edit-taxonomy now to fix different prefixes issue. So I should chang the prefix K__, in NCBI taxonomy file before merging it to the SILVA taxonomy file like that,

qiime rescript edit-taxonomy
--i-taxonomy ncbi-refseqs-taxonomy-unfiltered.qza
--p-search-strings 'k__Bacteria'
--p-replacement-strings 'd__Bacteria'
--o-edited-taxonomy ncbi-refseqs-edited-taxonomy-unfiltered.qza

I'd be more generic and simply do the following (just incase you have some Archaea... ):

qiime rescript edit-taxonomy \
    --i-taxonomy ncbi-refseqs-taxonomy-unfiltered.qza \
    --p-search-strings 'k__' \
    --p-replacement-strings 'd__' \
    --o-edited-taxonomy ncbi-refseqs-edited-taxonomy-unfiltered.qza

@Sabrin

Finally, Is there is a simple tutorial I can follow to export my files into R formats for further visualizations please.

Try to keep the questions on topic. Ideally, this question should be a separate post. But if you search this forum you'll be able to fins many examples and discussions.