Create.database for ASV

Xavi.Fonoll · February 4, 2022, 4:43am

Hello,

So far I have used create.database in MOTHUR to create a nice table with the different OTUs. I would like to do the same for ASV but I can’t succeed. When I enter the command, MOTHUR just does nothing.

Here is part of the log file:

mothur > 
remove.lineage(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.taxonomy, taxon=Chloroplast-Mitochondria-unknown-Eukaryota)

/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.accnos, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta)
Removed 377 sequences from your fasta file.
Removed 10872 sequences from your count file.

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table

/******************************************/

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.pick.taxonomy
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.accnos
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta


mothur > 
dist.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, cutoff=0.03)

Using 8 processors.

Sequence	Time	Num_Dists_Below_Cutoff

It took 2186 secs to find distances for 30748 sequences. 3399985 distances below cutoff 0.03.


Output File Names: 
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.dist

mothur > 
make.shared(count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table)

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.shared
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.list


mothur > 
classify.otu(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.pick.taxonomy, label=ASV)
ASV

Output File Names: 
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.cons.taxonomy
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.cons.tax.summary


mothur > 
get.oturep(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.list, fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, column=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.dist, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table)
You did not provide a label, using ASV.
ASV	30748

Output File Names: 
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.rep.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.rep.fasta


mothur > 
create.database(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.list, label=ASV, repfasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.rep.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.rep.count_table, constaxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.cons.taxonomy)

Is the problem due to the dist file I enter when I type the command get.oturep?

pschloss · February 8, 2022, 5:19pm

You shouldn’t need to run get.oturep or get distances since ASVs are generated without distances. Can you try using the current fasta file instead?

Pat

Xavi.Fonoll · September 14, 2022, 2:57pm

Hello,

I’m trying this again but I’m not succeeding. It seems that I need a repfasta file for this.

Here is my log file:

mothur >
remove.lineage(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.taxonomy, taxon=Chloroplast-Mitochondria-unknown-Eukaryota)

/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.accnos, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta)
Removed 503 sequences from your fasta file.
Removed 12506 sequences from your count file.

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table

/******************************************/

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.pick.taxonomy
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.accnos
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta

mothur >
make.shared(count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table)

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.shared
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.list

classify.otu(list= stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.list, count= stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table, taxonomy= stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.nr_v138_1.wang.pick.taxonomy, label=ASV)
ASV

Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.cons.taxonomy
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.cons.tax.summary

mothur >
create.database(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.list, label=ASV, fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table, constaxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.cons.taxonomy)
[WARNING]: fasta is not a valid parameter, ignoring.

pschloss · September 15, 2022, 4:23pm

The argument for create.database should be repfasta, not fasta. If you’re doing ASVs, then you’ll want to do get.oturep as you did earlier to generate stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.rep.fasta

Xavi.Fonoll · September 16, 2022, 5:23pm

Thank you Pat,

I ended up creating the database without the repfasta file.

mothur >
create.database(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.list, label=ASV, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table, constaxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.ASV.ASV.cons.taxonomy)

What I did after was to paste the info from the database output file into an excel sheet and I combined it with the data found in the files stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta and
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table

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Create.database for ASV

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