how can i get a reference-seqs in closeed-reference clusttering

i want analysis in to group of soil sample
so i amplified DNA in V3 and V4 and started analysis

but i have some metter to cluster DNA

qiime vsearch cluster-features-closed-reference
--i-table derep_table.qza
--i-sequences derep_seqs.qza
--i-reference-sequences sepp-refs-silva-128.qza
--p-perc-identity 0.99
--o-clustered-table table-dn99.qza
--o-clustered-sequences rep-seqs-dn99.qza
--o-unmatched-sequences unmatched-seq-dn99.qza

this code printed error
"Invalid value for '--i-reference-sequences': Expected an artifact of at
least type FeatureData[Sequence]. An artifact of type SeppReferenceDatabase
was provided."

i readed "OTU picking strategies"
if i use non-overlapping amplicon, like V2 and V4 in rRNA, i should use close-reference clustering....

but i dont know what is reference-sequence and how i get it?? i search in forum and google but i cant find this information...
please help me! thank you

Hi @svbreqwaiu01, the sepp-refs-silva-128.qza file is for fragment insertion. If you'd like to perform closed-reference OTU picking follow the instructions here. The reference files (GreenGenes and SILVA Sequence files) to use can be found on the Data resources page.

-Mike

@SoilRotifer
sorry and very thank you so much for your reply
is it your mean that i can closed-clustering with Marker gene reference databases??

As explained in the linked resources, this is exactly what closed-reference clustering means. You are only retaining those sequences that cluster within / match a given reference database within a defined percent similarity. The following article does a great job comparing some of these approaches:

Callahan, Benjamin J., Paul J. McMurdie, and Susan P. Holmes. 2017. “Exact Sequence Variants Should Replace Operational Taxonomic Units in Marker-Gene Data Analysis.” The ISME Journal 2 (12): 1–5. Exact sequence variants should replace operational taxonomic units in marker-gene data analysis | The ISME Journal

-Mike

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