Creating and Using a Custom HMM Database

When to use a custom HMM database

The PHROGs database covers the majority of known phage protein families, but it won't have entries for newly characterised or niche families. A custom HMM database lets you annotate those proteins alongside the standard PHROGs search.

Good candidates include:

• A set of structural proteins (e.g. tailspike variants, receptor-binding proteins) from a well-characterised phage genus not yet in PHROGs.
• A family of integrases, anti-CRISPR proteins, or any other group you have curated multiple sequence alignments (MSAs) for.
• Any group of proteins you want consistently flagged across a large annotation run.

The workflow is: collect FASTA MSAs (one file per family) → run pharokka create-hmm to build the .h3m profile database → pass it to pharokka run with --custom_hmm. Hits appear in pharokka_cds_final_merged_output.tsv under custom_hmm_id, and are included in the .gff and .gbk outputs as custom_annotation=....

Building the database

pharokka v1.4.0 allows you to input a custom HMM profile database using the --custom_hmm parameter.

The easiest way to do this is with pharokka create-hmm, which comes with pharokka:

pharokka create-hmm -i <input directory of MSAs> -o <outdir> -p <prefix>

The resulting output you need to feed into pharokka run with the --custom_hmm parameter will be outdir/prefix.h3m.

Input

pharokka create-hmm creates HMM profiles for each file in the input directory. It takes the name as the custom annotation for that HMM. It requires FASTA format MSAs (multiple sequence alignments) e.g.

>gene1
--------------------MERCMYDLSHFSFMTGRIGQLQTLTCIPVVSGDSFSVNFQGVFRLSPLRRNMVIDCMVDLFAFYVPYRHVYG-----DDWENFMLQGTDETVTFGTAD-FTTN-AVEYLGSQ-----R-TTGVAPLWRLASYNQIWNRYFRVPSDTSAILSNTALE---A-GVNKNKWGRYCARPKVIWSTGVDTTTD--AAD--REVTVTANQLDITDLDAVKGRYASEQRRDWFG-QRYNDILKNQFGGGAGTDADERPTLIMRKQHFLSGYDVDGTGDATLGQYSGKSAAVCDLQIPRKWFPEHGSL-WIMALLRFPTIHEEEIHYLFKKSQPTYDEISGDPDIWERKAPIEHQVQDFFTESTDTTSLGFMPYGQWYRYHPNVVHNQFTEVTGFSFVSA-RPT-----SKDTARYIQDDEYTPTFQTTSLGHWQSQARVDVEAVRPIPGPLKSIFAGV------------
>gene2
MSKSSGYATTSKPQEVVTRDIERKPYDLSHWSFKTGHIGRLQTFSVIPVVAGDSIELNMSAVLRLSPLRHFMYLDAVVDLFAFYVPHRHVYG-----SNWTAFLKAGVDEGSTLGTTT-FTGSNRPECLGVY-----FAAGQVLPSWSVHPYTMIWNNYFRDPQVDADAKTDATYLIGLTYPDPILKYGLPVCHLKRSWNTGVASGLS--TAD--YSLALSGGEVDLYQMSQLKGRLQTEQARDWFAFARYRDILKYTWDSEVNIDADQRPELIMRSTAWLSGKDVDGTDDATLGSYTGKSTGIFNLSFPSRFFAEHGSI-WIMGVVRFPPIVEAEAGYLNSKSEPTYKEISGDPSIIKHEPPISLNANEQFQG-ASSVDLGKIPYGQWYRESINMLSADYLEVSGHPFLLKSSIT-----SRATGVYVVPSQYDTVFSDTLMGHWNSQAFVDVRAKRFIPAPEDSIFAGTI-----------
>gene3
----------------MKGTDTRCLYDLDHWSHVSGNIGSLQTLSAIPVVAGDSMELNFTSLFRLSPLRRNLYLDAMVDLFAFYVPYRHVYG-----DTWIDFIKEGYDESQTLGTYT-IAAGEFINCTGAY-----LESQAVIPKWSIASYTRIWNRYFRHPTDSNEKDDDDLI----TASSGNQIYGYNCCHMKNIWSTGVDSELT--DDD--HKVDVTASKVDLLEIIQKKARFKTERQREWFG-QRYTDILDSVWGSNVNIDADERPELIMRTSTWLSGYDVDGTTETNIGTFSGKAFTTARLQFPMKYFNEHGTI-YIVALVRFPTIHAFERHYLFGKSEPTYKEIAGDPNVIRNEPPQAINPTDYIEN-ATATDVGLQPYAQWYRTHPSYTSLGFSNLDGHPFLQK-IIA-----NTDDAVYVDSNDYDEIFQTQQLQQWQSQGYVGLKAKRNIPDPRKSIFAGVK-----------

For example, if my input directory was called my_tail_msa and contained 3 MSA files named tail_family_1.fasta, tail_family_2.fasta and tail_family_3.fasta, the following command will create HMM profiles for all 3 MSA files, and combine them together into a h3m file with the prefix tail:

pharokka create-hmm -i my_tail_msa -o tail_hmms -p tail

The resulting file to be used with --custom_hmm is tail_hmms/tail.h3m.

Output

So on a genome, you would run pharokka as follows:

pharokka run -i <input fasta file> -o <output folder> -d <path/to/database_dir> -t <threads> --custom_hmm tail_hmms/tail.h3m

A gene hit to e.g. the tail_family_2 HMM profile found by PyHMMER will be indicated in the custom_hmm_id column of the pharokka_cds_final_merged_output.tsv output file as tail_family_2, with information about the custom_hmm_bitscore and custom_hmm_evalue from PyHMMER. The annotations will supplement any PHROG annotation found and will be included in the pharokka.gff and pharokka.gbk files as custom_annotation=tail_family_2 in this example.

usage: pharokka create-hmm [-h] [-i INDIR] [-o OUTDIR] [-p PREFIX] [-f] [-V]

pharokka create-hmm: Creates HMMs from FASTA formatted MSAs with PyHMMER for use with Pharokka v1.4.0 and higher.

options:
  -h, --help            show this help message and exit
  -i INDIR, --indir INDIR
                        Input directory containing FASTA formatted Multiple Sequence Alignments.
  -o OUTDIR, --outdir OUTDIR
                        Output directory to store HMM profiles.
  -p PREFIX, --prefix PREFIX
                        Prefix used to name HMMs. The relevant file will be 'prefix'.h3m
  -f, --force           Overwrites the output directory.
  -V, --version         Print pharokka Version