1. Download and install Anaconda, set Bioconda as well.

2. From ~/hb_database/hb_database_env.txt create the working environment.

    conda env create -file hb_database_env.txt
   

3. Init conda environment:

    conda activate hb_database
   

Download SwissProt uniprot_sprot.fa and create a local database.

	makeblastdb  \
	-in ~/db_uniprot/uniprot_sprot.fasta \
	-out ~/db_uniprot/ \
	-parse_seqids \
	-dbtype prot

Download Cdd database.

	makeprofiledb \
	-title Cdd \
	-in Cdd.pn \
	-out db_cdd \
	-threshold 9.82 \
	-scale 100.0 \
	-dbtype rps \
	-index true

Download Pfam-A.hmm & Pfam-A.hmm.dat and create a local Pfam database.

	hmmpress -f data/seqs/db_pfam/Pfam-A.hmm

Check here for set up steps.

Get from NCBI the .fasta file for the curated alpha subunits.

Only one alpha subunit per enzyme of interest.

	python 01_fetching_aa_seqs.py  \
	--input data/seqs/01_list_alpha_subunits.csv \
	--email <insert your email>

Input table example:

Do not use symbols such as (",", "/", etc.) in your input table. It may cause various parsing failures.

02_alpha_subunits_aa_sequences splits your multiple sequence 02_alpha_subunits_aa_sequences.fa file, and BLASTP each singular fasta file.

	python 02_do_blast.py \
	--input analyses/02_alpha_subunits_aa_sequences.fa  \
	--database data/seqs/db_uniprot/swiss_prot.fa

Check in the output xml files last/trailing entries; sometimes you can get eukaryotic genes

Parse .xml files and get UniProt reviewed and unreviewed (outgroup) sequences.

	python 03_get_blast_hits.py \
	--xml_in analyses/blast_outputs/separated_blast/

Nota Bene:

• separated_hits/interest (SwissProt reviewed sequences)
• separated_hits/outgroup (outgroup sequences)
• separated_hits/merged (reviewed + outgroup)

Get .fasta file for each hit.

	python 04_get_uniprot_fa.py \
	--input analyses/blast_outputs/separated_hits/

05_do_rps_blast.py uses .fa files in blast_outputs/separated_hits and outputs .cdd, .ko and .pfam tables.

	python 05_do_rps_blast.py \
	--input_fa analyses/blast_outputs/separated_hits/ \
	--db_cdd data/seqs/db_cdd/db_cdd \
	--evalue 0.01 \
	--db_pfam data/seqs/db_pfam/ \
	--db_ko data/seqs/db_ko/profiles/ \
	--ko_list data/seqs/db_ko/ko_list \
	--ko_config data/seqs/db_ko/config.yml

To enhance the performance of Ko annotation check .yml file.

Merge .cdd, .pfam, .ko tables in blast_outputs/separated_hits.

	python 06_create_annotated_table.py /
	--fasta_input analyses/blast_outputs/separated_hits/

Output example (ABE37059.1_Paraburkholderia_xenovorans_LB400.tsv):

Perform UniProt direct query.

Keyword implies exact gene name. Filtering implies deleting uncultured and fragments sequences.

	python 07_do_sw_query.py \
	--input_file data/seqs/01_list_alpha_subunits.csv

Get annotation for initial curated sequences (NBCI) analyses/blast_outputs/separated_fasta/ and SwissProt query sequences analyses/sp_query/

	python 05_do_rps_blast.py \
	-in analyses/blast_outputs/separated_fasta/ \
	-cdd data/seqs/db_cdd/db_cdd \
	-e 0.01 \
	-pfam data/seqs/db_pfam/ \
	-ko data/seqs/db_ko/profiles/ \
	-kl data/seqs/db_ko/ko_list \
	-kc data/seqs/db_ko/config.yml

	python 06_create_annotated_table.py /
	--fasta_input analyses/blast_outputs/separated_fasta/

	---------------------------------------------

	python 05_do_rps_blast.py \
	-in analyses/sp_query/ \
	-cdd data/seqs/db_cdd/db_cdd \
	-e 0.01 \
	-pfam data/seqs/db_pfam/ \
	-ko data/seqs/db_ko/profiles/ \
	-kl data/seqs/db_ko/ko_list \
	-kc data/seqs/db_ko/config.yml

	python 06_create_annotated_table.py /
	-in analyses/sp_query/

	python 08_do_format_seqs.py \
	--input_fa analyses/sp_query/

	python 08_do_format_seqs.py \
	--input_fa analyses/blast_outputs/

I would recommend to put .fa of analyses/sp_query/ in a separate directory

Keys: accession (NCBI accession #); sp_query (List of fasta files gathered after direct UniProt keyword query); filtered# (Number of SwissProt query sequences after filtration of fragments, etc.); unfiltered# (Number of SwissProt raw keyword query sequences); ncbi_ref (Manually curated NCBI files); sp_ref (Files resulted from RPS Blast); sp_ref# (Number of sequences in each RPS Blast file).

	python 09_prep_phylo.py \
	--initial_table data/seqs/01_list_alpha_subunits.csv \
	--ncbi_fa analyses/blast_outputs/separated_fasta/ \
	--sp_blast analyses/blast_outputs/separated_hits/merged/ \
	--sp_query analyses/sp_query/

Output: analyses/annotated_table.tsv

• Curated NCBI sequences can be found at analyses/blast_outputs/separated_fasta/
• SwissProt review sequences can be found at analyses/blast_outputs/separated_hits/relevant/
• SwissProt outgroup sequences can be found at analyses/blast_outputs/separated_hits/outgroup/
• SwissProt outgroup + reviewed sequences can be found at analyses/blast_outputs/separated_hits/merged/
• SwissProt keyword query sequences (filtered) can be found at analyses/sp_query/*.filtered
• Table with fasta files to be used further in the phylogenetic analyses analyses/annotated_table.tsv

Create analyses/phylo directory and put there merged .fasta files from analyses/blast_outputs/separated_fasta/, analyses/blast_outputs/separated_hits and analyses/sp_query/.

	python 10_phylo_env.py \
	--input_table analyses/annotated_table/annotated_table.tsv

Perform multiple alignment with MUSCLE. Use Trimal for alignment trimming (gap threshold of 0.05). Use IQ-TREE for phylogenetic tree rendering. Use UFboot option for bootstrapping.

In order to get accurate phylogenetic tree. Instead of GAMMA method use LG+R7 (default method in 11_generate_best_tree)

	python 11_generate_best_tree.py \
	--input analyses/phylo

Left join references titles for each alpha subunit in analyses/annotated_table.tsv

	python 12_get_references.py \
	--input_table analyses/annotated_table.tsv \
	--email <insert your email>

In order to annotate phylogenetic trees in analyses/phylo use R & ggtree (check the R script in /phylo/*.ipynb).

Use for jupyter notebook for .ipynb

Check /flow_charts for flow chart creation.

Check /analyses/phylo/*.Rmd for rendering final reports.

Final files can be accessed in analyses/phylo/*. Final .html reports can be accessed in reports/*.