Tutorial¶
Quick description¶
A more complete description of the method can be found in the Introduction.
OrphHCA is designed to detect conserved hydrophobic segments (called HCA-segments) on multiple sequence alignment (MSA).
The input of OrphHCA is a MSA fasta file. OrphHCA is actually distributed as two scripts. The main script orphHCA and an utilitary script filterOrphHCA.
The orphHCA script performs the external domain annotation and the HCA-segments search. Then, it selects the segments corresponding to domains based on their overlaps and their conservation in the MSA. Finally, the script produces a flat file with the domain positions and an hmm database file built with hmmbuild.
The filterOrphHCA script can be used to compare the created hidden markov models (HMMs) with models from other databases. The script uses the hhsearch tool to perform the comparison.
Getting started¶
First you will need to install OrphHCA. A complete documentation on how to install OrphHCA can be found in Installation.
Warning
As OrphHCA built the amino-acid sequences from the sequences of the MSA, non amino-acids characters [“*”, ”!”, ”.”, ”?”, “-“] are removed. Other characters in the sequences are kept.
Running orphHCA¶
Running orphHCA without specific parameters.
$ orphHCA -i examples/EOG7CPB12.fasta -o examples/EOG7CPB12 -w examples/EOG7CPB12/ -v --keep-fas
Two files are created: examples/EOG7CPB12.out and examples/EOG7CPB12.hmm.
The first file ‘’examples/EOG7CPB12.out’’ contains the list of domains
found in each protein. The format of the file follows the xdom syntax.
>FBgn0179134_Dsec_1 77213 61 orph_0 Nan # 12 65203 258 orph_1 Nan # 202 258288 328 orph_2 Nan # 287 328395 772 orph_3 Nan # 388 772>FBgn0241472_Dyak_1 78013 61 orph_0 Nan # 12 65...
Each protein entry starts with a fasta header correspoding to the name of the
protein sequence, for example FBgn0179134_Dsec_1 followed by a space character
and the length of the protein sequence, here 772 for the protein
FBgn0179134_Dsec_1.
The lines following the fasta header correspond to domain positions.
The line 13 61 orph_0 Nan # 12 65 is made of four required columns,
13 61 orph_0 Nan, and followed by two commented columns, 12 65.
The numbers 13 61 in the required columns correspond to the start and stop
positions of the domain, the position are inclusive and the first amino-acid
of the sequence starts at 1. The name orph_0 corresponds to the domain name
and can be shared between the proteins, the Nan correspond to the e-value
field of the xdom and should be ignored as no e-values are computed.
The two optional columns 12 65 correspond to the full length of the domain.
The final positions, 13 61, are computed based on the domain position
conservation between the sequences and the original HCA-domain annotation of the
protein sequences can be longer, 12 65 in this example.
As a matter of comparison the positions 13 61 can be seen as the alignment
position, ali columns, of the annotation produced by hmmscan and
the columns 12 65 as the envelop of the domain, env columns in
hmmscan results.
The second file examples/EOG7CPB12.hmm is an hmm file generated
from hmmbuilt. All the domain models are concatened in this file.
Running filterOrphHCA¶
Running filterOrphHCA:
$ filterOrphHCA -f examples/EOG7CPB12/kept_fasta/ -i examples/EOG7CPB12.hmm -w examples/filtering_EOG7CPB12/ -d pfamA_v27.0_22Oct13.hhm -c 50 -v -o examples/EOG7CPB12.filtered.dat
The program takes as an input the directory of the fasta files corresponding to
the previously created HMMs, examples/EOG7CPB12/kept_fasta/,
with an HMM databases corresponding to the fasta file,
examples/EOG7CPB12.hmm, a working directory,
examples/filtering_EOG7CPB12/ and an external database against
which the created models are compared, pfamA_v27.0_22Oct13.hhm.
The output file, examples/EOG7CPB12.filtered.dat is a tab
delineated flat file of four columns.
model_name_1 target_name_1 similarity database_of_the_target_1model_name_1 target_name_2 similarity database_of_the_target_2...model_name_2 target_name_1 similarity database_of_the_target_1...
- All the targets having a similarity score strictly above the cutoff parameter,
-c 89, are reported.
Parameters of orphHCA¶
Required parameters¶
-
-i,--input: FILE¶ the MSA input file
-
-o,--output: FILE PREFIX¶ output file prefix (<output>.out : list of domains, <output>.hmm : hmmdatabase)
-
-w,--workdir: DIR¶ working directory
Optional parameters¶
-
-d,--database¶ list of the domain hmm databases to use
-
-s,--seqdb¶ path to the sequence database used for enrichment
-
-c,--core¶ number of cores to use; default=1
-
--perc-hca¶ minimal percentage of sequences in the MSA that should have a domain , default=20
-
--nb-hca¶ minimal number of sequences in the MSA that should have a domain
-
--perc-over,default=80¶ minimal percentage of overlap allowed between hca segment for them to be considered as part of the same domain
-
--nb-over¶ minimal number of overlapping amino-acids between two hca segments to consider them as the same
-
--hca-size¶ minimal size to consider a hca segment as a domain, default=30
-
--perc-hmm¶ maximal percentage of overlap allowed between a hca segment and a hmm domain , default=0
-
--nb-hmm¶ maximal number of overlapping amino-acids allowed between an hca segment and an hmm domain
-
--keep-fas¶ keep fasta results, fasta alignment are needed by hhsearch in the filtering program
-
-v,--verbose¶ active/inactive verbose mode
Parameters of filteringOrphHCA¶
Required parameters¶
-
-f,--fastadir¶ the directory with fasta alignments
-
-i,--inputfile¶ the hmm database corresponding to the fasta alignments
-
-w,--workdir¶ the working directory
-
-d,--database¶ the list of hmm database to which the fasta alignments are compared to
-
-o,--output¶ the list of model that are similar to an other model in a database
-
-c,--cutoff¶ the similarity cutoff