-
Notifications
You must be signed in to change notification settings - Fork 1
Module 0 Geneious for microbial ecology
- Click Tools > Add/Remove Databases > Setup BLAST services
- Click Edit Databases
- Create new database with the +Add button
Database name: rRNA_typestrains/prokaryotic_16S_ribosomal_RNA
Display name: 16S_Type_strains
Description: 16S_Type_strains
tick type - nucleotide
> Tools >Preferences -- Tab Plugins&Features
mafft
fasttree
optionally you can get other phylogenetic tools such as PHYML, RAxML, MrBayes
If you drag and drop between folders, sequences are usually moved. If you want to copy sequences you can hold the Ctrl (German keyboards Strg) key wile drag and dropping.
> File > Import > from File
You can find the files in the owncloud folder...
If you are directly processing Sanger data e.g. from the ABI machine inhouse, just select all .abi files by entering *.abi
select all files you want to assemble
> Align/Assemble > De novo Assemble
Check the box on top - assemble by XX part of the name separated by _/- make your choice according to the sequence names e.g. with sequences looking like 'SYLT17-66_3FNem_MSA_7656_E09_2018-01-18.ab1' you would choose assemble by 1st part of the name separated by and _Underscore - the assembled sequences would then be called SYLT17-66.
Check all boxes in the RESULTS section. In the trim before assembly section, you should select 'retrim' to make sure you feed trimmed sequenced to the assembly process. You can check the trimming options - you want to trim from 3' and 5' and with a maximum error 0.005. If you have sequences that come from cloning inserts, you might also want to screen for cloning vector contamination.
Your assembled consensus sequences will be in the subfolder in a sequence list called 'Consensus Sequences'. If you have leftover reads that were not assembled, you can find them in a sequence list called 'Unused Reads'.
import from file
download from NCBI e.g. using the Nucleotide database and keyword searches
get hits from BLAST search - for type strains, use type strains database, for environmental seqs you can use nr
Include an outgroup in your analysis if you want to be able to root the tree! An outgroup should ideally be not too far away, but also distinct enough. One way to find such a set of sequences is to get representatives from the taxonomic level ABOVE your analysis - e.g. If you are working with Thiosymbion - they are a genus level clade in the Chromatiaceae family, get sequences from other genera in the family Chromatiaceae such as Marichromatium, Thiocapsa ...
select sequences for analysis - references and your target sequences
use mafft to align sequences
> Align/Assemble > Multiple Align > MAFFT Alignment\
- check 'automatically determine sequences direction' to make sure that orientation is OK
fasttree gives quick tree for overview
> Tree > FastTree
choose GTR and Gamma20 optimization
root tree with your outgroup
choose your tip labes - right hand panel 'show tip labels' - you can select more than one with keeping Ctrl pressed while clicking, interesting parameters could be 'isolation source', 'host', or taxonomy