###### tags: `Nanopore Sequencing`

# Prokaryotic Genome Annotation Pipeline (PGAP) - Annotation with NCBI Pipeline

## Create the Input files

### 1. FASTA file

Each sequence in the file must have a definition line beginning with '>' and a unique identifier (SeqID), eg >contig_1 or >contig_2.

e.g.: nord1.fasta

WITH:

1. Chromosome
>contig_1 Escherichia coli nord1, complete genome [completeness=complete] [topology=circular] [location=chromosome]
ATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTAAGGCCGCCAGGCAAATTCTGTTTCATCAGACCGCTTCTGCGTTCTGATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACATCTTCGGCGTTGTAAGGTTAAGCCTCACGGTTCATTAGTACCGGTTAGCTCAACGCATCGCTGCGCTTACACACCCGGCCTATCAACGTCGTCGTCTTCAACGTTCCTTCAGGACTCTCAGGGAGTCAGGGAGAACTCATCTCGGGGCAAGTTTCGTGCTTAGATGCTTTCAGCACTTATCTCTTCCGCATTTAGCTACCGGGCAGTGCCATTGGCATGACAACCCGAACACCAGTGATGCGTCCACTCCGGTCCTCTCGTACTAGGAGCAGCCCCCTCAGTTCTCCAGCGCCCACGGCAGATAGGGACCGAACTGTCTCACGACGTTCTAAACCCAGCTCGCGTACCACTTTAAATGGCGAACAGCCATACCCTTGGGACCTACTTCAGCCCCAGGATGTGATGAGCCGACATCGAGGTGCCAAACACCGCCGTCGATATGAACTCTTGGGCGGTATCAGCCTGTTATCCCCGGAGTACCTTTTATCCGTTGAGCGATGGCCCTTCCATTCAGAACCACC.....
2. Plasmids
>contig_2 Escherichia coli nord1, complete genome [completeness=complete] [topology=circular] [location=plasmid] [plasmid-name = pNORD1_2]
ATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCGGCGCTACGGCGTTTCACTTCTGAGTTCGGCATGGGGTCAGGTGGGACCACCGCGCTAAGGCCGCCAGGCAAATTCTGTTTCATCAGACCGCTTCTGCGTTCTGATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACATCTTCGGCGTTGTAAGGTTAAGCCTCACGGTTCATTAGTACCGGTTAGCTCAACGCATCGCTGCGCTTACACACCCGGCCTATCAACGTCGTCGTCTTCAACGTTCCTTCAGGACTCTCAGGGAGTCAGGGAGAACTCATCTCGGGGCAAGTTTCGTGCTTAGATGCTTTCAGCACTTATCTCTTCCGCATTTAGCTACCGGGCAGTGCCATTGGCATGACAACCCGAACACCAGTGATGCGTCCACTCCGGTCCTCTCGTACTAGGAGCAGCCCCCTCAGTTCTCCAGCGCCCACGGCAGATAGGGACCGAACTGTCTCACGACGTTCTAAACCCAGCTCGCGTACCACTTTAAATGGCGAACAGCCATACCCTTGGGACCTACTTCAGCCCCAGGATGTGATGAGCCGACATCGAGGTGCCAAACACCGCCGTCGATATGAACTCTTGGGCGGTATCAGCCTGTTATCCCCGGAGTACCTTTTATCCGTTGAGCGATGGCCCTTCCATTCAGAACCACC....

### 2. YAML files
Both *.yaml files must have these structures. You can find an example for each *.yaml on server here:

$:/mnt/data_1/pgap_ncbi/test_genomes-2020-09-24.build4894/MG37

#### a) input.yaml

fasta:
  class: File
  location: nord1.fasta
submol:
  class: File
  location: submol.yaml

#### b) submol.yaml

topology: circular
comment: 'There is no really a biologist Arnold Schwarzenegger'
consortium: 'SkyNet consortium'
sra:
    - accession: 'ERR123456789'
tp_assembly: true
organism:
    genus_species: 'Escherichia coli' 
    strain: 'Nord1'
contact_info:
    last_name: 'Braun'
    first_name: 'Sascha'
    email: '[e-mail]'
    organization: 'Institute of Klebsiella foobarensis research'
    department: 'Department of Using NCBI'
    phone: '301-555-0245'
    street: '1234 Main St'
    city: 'Docker'
    postal_code: '12345'
    country: 'Lappland'
    
authors:
    -     author:
            first_name: 'Arnold'
            last_name: 'Schwarzenegger'
            middle_initial: 'T'
    -     author:
            first_name: 'Linda'
            last_name: 'Hamilton'
bioproject: 'PRJNA9999999'
biosample: 'SAMN99999999'      
# -- Locus tag prefix - optional. Limited to 9 letters
locus_tag_prefix: 'nord01'
publications:
    - publication:
        pmid: 16397293
        title: 'Discrete CHARMm of Klebsiella foobarensis.'
        status: published  # this is enum: controlled vocabulary
        authors:
            - author:
                first_name: 'Martin'
                last_name: 'Reinicke'                
            - author:
                  first_name: 'Sascha'
                  last_name: 'Braun'
                  middle_initial: 'D'

## Start PGAP

1. copy all needed files (fasta, input.yaml, submol.yaml) into one folder

e.g.:
$:/mnt/data_1/pgap_ncbi/dresi_ref/nord1

2. navigate to folder:

$:/mnt/data_1/pgap_ncbi

3. start ./pgab.py

$:./pgap.py -r -o dresi_ref/nord1/nord1_results dresi_ref/nord1/input.yaml

-o outdir directory (will automatically written)
-r Report to NCBI whenever the pipeline is run

Usefull commands

-r, --report-usage-true	Report to NCBI whenever the pipeline is run.
-n, --report-usage-false	Do not report to NCBI.
-o path, --output path	Output directory to be created, which may include a full path.
--no-internet	Disable internet access for all programs in pipeline.
--taxcheck	Also calculate the Average Nucleotide Identity to type assemblies
--taxcheck-only	Only calculate the Average Nucleotide Identity to type assemblies, do not run PGAP