vsearch open-reference pipeline and phylogenetic tree

[Lu_Zhang]

Hi

My commands as follow:

qiime tools import \
  --input-path $2/seqs.fna \
  --output-path $2/seqs.qza \
  --type 'SampleData[Sequences]'


# import reference database gg13_8 
qiime tools import \
  --input-path gg_13_8_otus/rep_set/97_otus.fasta \
  --output-path $2/97_otus.qza \
  --type 'FeatureData[Sequence]'


# dereplication 
mkdir -p $2/dereplicated
qiime vsearch dereplicate-sequences \
  --i-sequences $2/seqs.qza \
  --o-dereplicated-table $2/dereplicated/table.qza \
  --o-dereplicated-sequences $2/dereplicated/rep-seqs.qza

# clustering 97% OTU
 
mkdir -p $2/clustered 

# open reference 

qiime vsearch cluster-features-open-reference \
  --i-table $2/dereplicated/table.qza \
  --i-sequences $2/dereplicated/rep-seqs.qza \
  --i-reference-sequences $2/97_otus.qza \
  --p-perc-identity 0.97 \
  --o-clustered-table $2/clustered/table-or-97.qza \
  --o-clustered-sequences $2/clustered/rep-seqs-or-97.qza \
  --o-new-reference-sequences $2/clustered/new-ref-seqs-or-97.qza \
  --p-threads 48

# remove chimera - open reference 

# 1. run de novo chimera checking 
qiime vsearch uchime-denovo \
  --i-table $2/clustered/table-or-97.qza \
  --i-sequences $2/clustered/rep-seqs-or-97.qza \
  --output-dir $2/uchime-dn-out

# 2. visualize chimera result 

qiime metadata tabulate \
  --m-input-file $2/uchime-dn-out/stats.qza \
  --o-visualization $2/uchime-dn-out/stats.qzv

# 3. Exclude chimeras but retain “borderline chimeras

qiime feature-table filter-features \
  --i-table $2/clustered/table-or-97.qza \
  --m-metadata-file $2/uchime-dn-out/chimeras.qza \
  --p-exclude-ids \
  --o-filtered-table $2/uchime-dn-out/table-nonchimeric-w-borderline.qza

qiime feature-table filter-seqs \
  --i-data $2/clustered/rep-seqs-or-97.qza \
  --m-metadata-file $2/uchime-dn-out/chimeras.qza \
  --p-exclude-ids \
  --o-filtered-data $2/uchime-dn-out/rep-seqs-nonchimeric-w-borderline.qza

Q1: Finally, with 159 samples, I have 74219 OTUs. Is that too much?
I searched the forum with a similar question: Denoising vs OTU picking methods - #5 by Dchung. In this post, he has 80000, which is too high, so i am thinking if vsearch open-reference pipeline reliable?

Q2: Can I do downstream diversity analysis by using greengenes' trees?
I have read a related questions about how to build phylogenetic tree after using vsearch otu-clustering method, I searched the posts in forum with this similar question :

Error when using sepp with vsearch clustered rep-seqs

Yes that is expected, but both are unnecessary after using closed-reference OTU clustering, since you should just use the greengenes reference tree instead of trying to build your own. I am a rookie so actually don’t know how to use the greengene reference after closed-reference OTU to generate tree.qza. So I would be really grateful if you could give me a clue. Could you please re-run sepp with the --p-debug option, and share the output here? Thanks. I run the command with --p-debug op…

In this question, it mentioned that trees from greengenes database can be directly used if closed-reference is used. However in my case, I used open-reference, but I didn't use the de novo seqs, can I directly use the trees from greengenes?

Thank you so much for helping!

Best,
Lu

[andrewsanchez]

Hi @Lu_Zhang!

@Lu_Zhang

However in my case, I used open-reference, but I didn’t use the de novo seqs, can I directly use the trees from greengenes?

Have you seen the OTU Clustering Tutorial?

[Lu_Zhang]

Yes. I read the tutorial and I already got the feature table and representative sequence by following it. Now I wanna use this result to do downstream analysis like using unifrac distance to see beta diversity. However in this case I need to construct a tree first. Since even though I used the open reference I got some de novo seqs, but I only used the ones based on reference,it’s like close reference.(open reference is combination of de novo and close reference. The reason I used open reference is I am thinking maybe in sometime I might be interested in these de novo seqs and then I could go back to check these de novo seqs) So I am wondering if I can use the green genes its own tree. Thank you so much for your reply!

[cherman2 (Chloe Herman)]

Hello @Lu_Zhang
Sorry for our late response. I want to preface this by saying that I am not 100% sure if I am answering your question. If I don’t answer your question, please let me know and I will try my best to give a better answer next time
I think you might be able to use sepp-insertion and use the green genes reference tree if you want.
This is an article that talks about using open reference and creating phylogenies

Phylogenetic Placement of Exact Amplicon Sequences Improves Associations with...

Here is a qiime2 doc that has the green genes reference for sepp insertion
https://docs.qiime2.org/2020.8/data-resources/#sepp-reference-databases
Thank you for your patience.