SOP for ITS analysis using Sanger sequencing?

jhines1 · January 2, 2019, 6:27pm

Sorry if this is a ridiculous question, but I am completely new to bioinformatics & relatively new to molecular biology, coming from a more traditional Ecology background. I have fungal ITS sequences produced from the Sanger method in an experimental design using leaf discs incubated in microcosms & I am now ready to start analyzing the community.

I’ve noticed that there are SOP’s for 454, Illumina, & other sequencing platforms, but I have yet to find any SOP for sequences produced using the Sanger method. Does an SOP exist for this? If not, what is the best approach to take when coming up with a procedure?

Again, sorry if this is an elementary question, but I’m definitely learning “on-the-fly”.

Cheers!

svazquez · January 3, 2019, 12:00am

Hi!
You mentioned “analyzing the community”… did you sequenced a clone library constructed after amplification of ITS sequences from a total DNA extracted from an environmental sample? or are you talking about ITS sanger sequences obtained from isolates?
If the Sanger ITS sequences come from clone libraries obtained from environmental samples, I guess you can use (adapt) the 454 SOP (I did it with 16S bacterial clone libraries) but you will have to see which database you can use to identify OTUs.
But, if you have ITS sequences obtained from isolates, then you will have to deal one by one if you want good quality analysis and reliable identification. In that case, I guess nothing to do with Mothur, but I may be able to help. Just let me know!
Cheers,
Susana

jhines1 · January 3, 2019, 12:59am

Hey Susana & thanks for your reply!!

Yes, I sequenced the ITS2 region from an environmental sample (leaf discs in a microcosm incubated with natural stream water). I will look into your recommendations & try to see how I could/should modify the protocol. It seems as since they are ITS sequences (instead of 16S sequences), it may not be as straight-forward. After I posted this, I found this question/answer on the forum. I definitely have more homework to do!! But, I will definitely look more into it & see exactly what the differences are & proceed from there.

Thanks again for your response!

Kendra · January 3, 2019, 5:30pm

here’s an example of how to analyze sanger sequenced ITS, it’s several years old so some of the command defaults may have changed.
https://mothur.org/wiki/Forest_soil_community_analysis

you could also look at my batch file for miseq ITS, which is different data but should have current commands/options

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

jhines1 · January 3, 2019, 6:40pm

Hey Kendra & thanks for the reply! I found the forest soil tutorial late last night in the wiki & I’ll be working through it today or tomorrow. Also,thanks for the MiSeq code!! I sincerely appreciate your help.

system · January 13, 2019, 6:40pm

This topic was automatically closed 10 days after the last reply. New replies are no longer allowed.

Topic		Replies	Views
Classification of Illumina MiSeq and Sanger sequences into same OTUs Theory behind mothur	2	403	February 28, 2021
ITS SOP and ITS databases Theory behind mothur	4	2428	October 2, 2017
analysing fungal 454 sequences Commands in mothur	8	6200	August 13, 2015
Unclassified fungi (Fungal analysis ITS2 region) Theory behind mothur	15	2003	June 29, 2020
Towards an SOP for fungal ITS sequences	9	1320	April 14, 2022

SOP for ITS analysis using Sanger sequencing?

Related Topics