diff --git a/wolf_tutorial.md b/wolf_tutorial.md index 7e898df..909a780 100644 --- a/wolf_tutorial.md +++ b/wolf_tutorial.md @@ -27,7 +27,7 @@ The new database system used by the OBITools3 (called **DMS** for Data Managemen Any hybrid of those 2 works too. * View names must be unique within a DMS, in other words, views can not be overwritten. - + * All tools accept different input and output DMS. * If the output DMS is not given, the input DMS is used. @@ -70,7 +70,7 @@ Download this archive containing the reads and the ngs file: And unzip it: - tar -zxvf wolf_tutorial.tar.gz + tar -zxvf wolf_tutorial.tar.gz 1. Import the first set of reads, with : @@ -139,11 +139,28 @@ Unlike the OBITools1, the OBITools3 make it possible to run ngsfilter before ali #### Build a reference database -1. Download the sequences, or rather, just the files with mammal sequences for this tutorial: +Building the reference database is costly in time and disk space so you can simply download this already built one: + +[v05_refs.fasta.gz](/uploads/fe71e2e103014d70a1bf9307e377ce2b/v05_refs.fasta.gz) + +With the associated taxdump: + +[taxdump.tar.gz](https://drive.google.com/file/d/1lMV5PWg122ZmhQtx0iq5m5iUKvVBZMUC/view?usp=sharing) + +And import them (note that you could import them in another DMS): + + obi import v05_refs.fasta.gz wolf/v05_refs + obi import --taxdump taxdump.tar.gz wolf/taxonomy/my_tax + +You can then resume at the next part "**Clean the database**". + +Otherwise, to build the database yourself from the start: + +1. Download the sequences (except human and environmental samples): mkdir EMBL cd EMBL - wget -nH --cut-dirs=5 -Arel_std_mam_\*.dat.gz -m ftp://ftp.ebi.ac.uk/pub/databases/embl/release/std/ + wget -nH --cut-dirs=5 -A rel_std_*.dat.gz -R rel_std_hum_*.dat.gz,rel_std_env_*.dat.gz -m ftp://ftp.ebi.ac.uk/pub/databases/embl/release/std/ cd .. 2. Import the sequences in the DMS: @@ -164,23 +181,33 @@ For EMBL files, you can give the path to a directory with several EMBL files. 5. Use ecoPCR to simulate an *in silico* PCR with the V05 primers: - obi ecopcr -e 3 -l 50 -L 150 -F TTAGATACCCCACTATGC -R TAGAACAGGCTCCTCTAG --taxonomy wolf/taxonomy/my_tax wolf/embl_refs wolf/v05_db + obi ecopcr -e 3 -l 50 -L 150 -F TTAGATACCCCACTATGC -R TAGAACAGGCTCCTCTAG --taxonomy wolf/taxonomy/my_tax wolf/embl_refs wolf/v05_refs #### Clean the database 1. Filter sequences so that they have a good taxonomic description at the species, genus, and family levels: - obi grep --require-rank=species --require-rank=genus --require-rank=family --taxonomy wolf/taxonomy/my_tax wolf/v05_db wolf/v05_db_clean + obi grep --require-rank=species --require-rank=genus --require-rank=family --taxonomy wolf/taxonomy/my_tax wolf/v05_refs wolf/v05_refs_clean -2. Build the reference database specifically used by the OBITools3 to make ecotag efficient: +2. Dereplicate identical sequences: + + obi uniq --taxonomy wolf/taxonomy/my_tax wolf/v05_refs_clean wolf/v05_refs_uniq + +3. Ensure that the dereplicated sequences have a taxid at the family level: + + obi grep --require-rank=family --taxonomy wolf/taxonomy/my_tax wolf/v05_refs_uniq wolf/v05_refs_uniq_clean + + +4. Build the reference database specifically used by the OBITools3 to make ecotag efficient: + + obi build_ref_db -t 0.97 --taxonomy wolf/taxonomy/my_tax wolf/v05_refs_uniq_clean wolf/v05_db_97 - obi build_ref_db -t 0.95 --taxonomy wolf/taxonomy/my_tax wolf/v05_db_clean wolf/v05_db_definitive #### Assign each sequence to a taxon Once the reference database is built, taxonomic assignment can be done using the `ecotag` command: - obi ecotag -m 0.95 --taxonomy wolf/taxonomy/my_tax -R wolf/v05_db_definitive wolf/cleaned_sequences wolf/assigned_sequences + obi ecotag -m 0.97 --taxonomy wolf/taxonomy/my_tax -R wolf/v05_db_97 wolf/cleaned_sequences wolf/assigned_sequences ### 8. After the taxonomic assignment @@ -216,7 +243,7 @@ or convert the dot file to a png image file: You will get something like this: -![wolf](/uploads/7cb6d08cd5c645ee20ac49d233d2dc9a/wolf.png) +![wolf](/uploads/12ce2dd47456baa59fcfa3d09df2643c/wolf.png) #### Export the results