Analisis of FUNGI with UNITE database in Mothur

sara.dan · September 13, 2017, 7:08am

Hi all,
I’m a PhD student, just at the begin with mothur analysis… I got a problem when I have to align sequences usign “align.seqs” comand.

I downloaded the database of fungi from UNITE website, but I didn’t find the “aligned file”.

I tried to process align.seqs comand with all file found in the UNITE directory dowloaded, as:
UNITEv6_sh_97.fasta
UNITEv6_sh_99.fasta
UNITEv6_sh_dynamic.fasta

But none is working…please can you help me?
Any help will be very apreciate

best regards to all

Sara

pschloss · September 14, 2017, 11:28am

Assuming you have 18S rRNA gene sequence data, you would probably want the eukaryotic silva database that we have posted

https://www.mothur.org/wiki/Silva_reference_files

Pat

sara.dan · September 14, 2017, 12:48pm

Hi Pat, thank for replying.

I obtained sequences from 18S-ITS1-5.8S regions of fungi, and now I’m trying to process data with Mothur!

I thought that in UNITE folder there was the align.file and when I didnt find it I was in doubt!

Regards

Kendra · September 14, 2017, 3:03pm

ITS can’t be aligned across all fungi because there’s too much evolution. Here is my batch for processing ITS2 seqs with mothur.

github.com

krmaas/bioinformatics/blob/master/mothur.fungal.batch

###################################################################
###################################################################
###################################################################


##Basic mothur processing of MiSeq sequences using ITS2 primers.  This batch is based on mothur.org/wiki/MiSeq_SOP  All commands are explained in much more detail in that SOP. If your data is not demultiplexed, see the chunk of code at the bottom of this file.
##I use a computer with a high memory node (512gb RAM) with 32 processors. You'll need to adjust processors used to match your system. 


#############
##### I've started using basemount to download the fastq directly to the server. This will download and rename the files to include sample name and sample ID.
#p=PROJECTNAME
#mkdir -p $p/fastq
#for f in basespace/Projects/$p/Samples/*/Files/*.gz; 
#do s=${f##basespace/Projects/$p/Samples/}; s=${s%%/*}; 
#cp $f $p"/fastq/"$s"."${f##*Files/}; 
#done
#####################

### In addition to your sequence files and the oligos file, you need some files from the mothur website to be accessible (either in your path or in the folder you are working in). The line below moves them from my public directory on the BBC server to your current directory.

This file has been truncated. show original

I’ve never worked with ITS1 data so don’t know how well UNITE covers that region, you may be better off with Silva

sara.dan · September 18, 2017, 7:26am

Hi Path,

Really thank for your recommendation!! We have also primers to amplify the ITS2 region, but as we are at the begin of fungi analysis, I’m trying to test different software package in order to understand if I will be able to work with fungi

Thank again!

sara

sara.dan · September 18, 2017, 7:27am

Hi Patrick,

Really thank for your recommendation!! We have also primers to amplify the ITS2 region, but as we are at the begin of fungi analysis, I’m trying to test different software package in order to understand if I will be able to work with fungi

Thank again!

sara

sara.dan · September 18, 2017, 7:27am

Hi Patrick,

Really thank for your recommendation!! We have also primers to amplify the ITS2 region, but as we are at the begin of fungi analysis, I’m trying to test different software package in order to understand if I will be able to work with fungi

Thank again!

sara

sara.dan · October 31, 2017, 11:41am

Dear Patrick,

When I run a command I get a Logfile; but every time I run the same command again, the previous logfile is deleted…

Is it normal?

Kendra · October 31, 2017, 3:44pm

are you renaming the logfiles? they should be named with a time stamp which makes each filename unique

sara.dan · October 31, 2017, 3:50pm

I’m not renaming the logfiles actually. I will do it from now!

Thanks!!

sara.dan · October 31, 2017, 5:02pm

It works! Now I have more than one logfile!
just last question… I’m trying to analyse 16S using this procedure but mothur stop all time at the command classify.seqs

#unique.seqs(fasta=current)

#count.seqs(name=current.names)

#align.seqs(candidate=current.fasta, template=\SILVA_128_mothur\silva.seed_v128.align)

#filter.seqs(fasta=current)

#unique.seqs(fasta=current.unique.filter.fasta, name=current.names)

#pre.cluster(fasta=current.unique.filter.unique.fasta, name=current.names)

#Chimera.vsearch procedure:
vsearch --derep_fulllength
vsearch --cluster_fast
vsearch --sortbysize
vsearch --uchime_denovo
*Using fasta.file, obtained by vsearch --uchime_denovo, I try to run the classify.seqs command.

#classify.seqs(fasta=current, reference=\SILVA_128_mothur\silva.seed_v128.align, taxonomy=SILVA_128_mothur\SEED\silva.seed_v128.tax, output=simple)
*The command is not working, because mothur stop and close the run.

In the logfile there is no explanation about why mothur is closing, and I don’t understand why…

Can you help me?

Really thank for your help…

Best regards

Kendra · November 1, 2017, 4:20pm

do you get any errors? you’re telling it to look in 2 different locations for your reference and taxonomy, are they really in different folders?

Topic		Replies	Views
analysing fungal 454 sequences Commands in mothur	8	6201	August 13, 2015
UNITE Database Theory behind mothur	3	7892	November 11, 2014
[ERROR]: template is not aligned, aborting in mothur454 analysis Commands in mothur	2	1175	March 14, 2016
formatting database into mothur format Commands in mothur	7	3720	September 14, 2016
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Analisis of FUNGI with UNITE database in Mothur

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