TITLE: Annotation for Fungi
AUTHORS: Sebastian Bassi, Virginia Gonzalez, Tristan Yang

[DESCRIPTION]
Protocol to annotate a fungi genome

[STEPS]
SECTION: Setup
1. Install Docker

If you don't have Docker already, install it. There are two versions, Docker Engine (also known as CE) and Docker Desktop. The Desktop version is more user friendly but since may require commercial license for large enterprise, this tutorial is based on the Docker engine. Both version will work in this protocol. Linux users can install both Docker CE and Desktop, while macOS and Windows users should install Docker Desktop.

Follow the installation instructions from https://docs.docker.com/engine/install/ 

SECTION: Run sspace_longread
4. Run the following command (replace /your_dir for the base directory where you have your data

 

SECTION: Run BWA Index
6. Run the following command (replace /your_dir for the base directory where you have your data

 

SECTION: Setup
2.  Get your data ready

You will need fastq data (long reads), short reads, and the assembly data. In the following code, the assembly data file is called assembly.fasta. The long reads file is called ID.fastq. The short reads should be two files (ID_R1.fastq.gz and ID_R2.fastq.gz). 
If you have more files for short reads, you can concatenate them so you end up with 2 files. For example, if you have ID_L001_R1.fastq.gz, ID_L002_R1.fastq.gz, ID_L001_R2.fastq.gz, ID_L002_R2.fastq.gz, you can concatenate them with these commands:

 

All files should be inside a directory, for example: your_dir
Inside your_dir there should be three directories: funannotate_prep, funannotate and funannotate/ipsout.
You can create them with this command:

 

SECTION: Run Gapcloser
5. Run the following command (replace /your_dir for the base directory where you have your data

 


SECTION: Run fastp
7. Run the following command (replace /your_dir for the base directory where you have your data)

 

SECTION: Run BWA mem
8. Run the following command (replace /your_dir for the base directory where you have your data). Replace CPU for your CPU count.

 

SECTION: Run SAMTOOLS
9. SAMTOOLS View, Sort and Index

Run the following command (replace /your_dir for the base directory where you have your data).

 

SECTION: Pilon
10. Run the following command (replace /your_dir for the base directory where you have your data).

 

SECTION: Funannotate
11. Funannotate Clean and Sort

Run the following command (replace /your_dir for the base directory where you have your data).

 

SECTION: RepeatMasker
12. Run the following command (replace /your_dir for the base directory where you have your data). Remember that is step requires the dfam38_full.0.h5 database installed in /your_dir

 


SECTION: Fuannotate Predict
13. Run the following command (replace /your_dir for the base directory where you have your data). Replace CPU for your CPU count.

 

SECTION: Interproscan
14. Run the following command (replace /your_dir for the base directory where you have your data). Replace CPU for your CPU count.

 


SECTION: Funannoate annotate
15. Run the following command (replace /your_dir for the base directory where you have your data)

 

SECTION: Setup
3. Download FamDB HDF5 database, Interproscan database and GeneMark license 


SECTION: Setup
3.1. FamDB HDF5 database

FamDB HDF5 database is needed for the RepeatMasker step. This database is partitioned by taxonomic groups, the partition needed for Fungi is partition number 0, for more information about partitions read this file:  
FamDB HDF5 database can be downloaded from here.
Bash commands to download, unzip and mv the database to /your_dir:

 

SECTION: Setup
3.2. Interproscan database

This DB is needed for the Interproscan step.
Download the Interproscan DB from here (this file is >5Gb).

Commands to download and untar:
 

SECTION: Setup
3.3. If you don't have a GeneMark license, get it from this page. License key file should be named gm_key and located in /your_dir. This license is need to run the Funannotate Predict step.