Alternative splicing (AS) is an important process of gene regulation at transcriptional level and substantially contributes to the understanding of proteomic diversity and function. In order to advance in large-scale research about the functional impact of AS on the proteome, automated methods are required for identifying AS events and linking them to functional regions of proteins in a systematic manner.
AstaFunk is a JAVA tool to study how diversity of a custom transcriptome translates into functional variation, based on standard transcriptome annotations and protein family profiles. In a nutshell, ASTAFUNK translates alternatively spliced parts of open reading frames (given by GTF annotation) on the fly into amino acid sequences. Subsequently, profile HMMs of Pfam database are searched against these amino acid sequences only in the regions of alternative splicing events. ASTAFUNK algorithm is designed to avoid redundant sequence scans in AS-enriched transcriptomes.
This document presents the information to download binaries, build AstaFunk from source code and execute basic commands.
Alternatively, the current version can always be obtained from the GIT repository. Clone the Git repository of the barna. The Barna library consists of a set of tools bundled with the package.
Build the binaries of AStalavista and create a distribution version.
Enter into the distribution directory and extract the files (.tgz or .zip). In barna.astalavista directory:
The current bundle uses 'astalavista' as the default tool. You can switch tools with the -t option and get help for a specific tool with -t <toolname> --help. This will print the usage and description of the specified tool
You will see:
|[--hmm <HMM_FILE>]||Profile-HMM file.|
|[--gtf <GTF>]||Gene annotation (GTF)|
Path to the directory with the genomic sequences, i.e., one fasta file per chromosome/scaffold/contig with a file name corresponding to the identifiers of the first column in the GTF annotation
|[(-r|--reference) <REFERENCE_FILE>]||Path to the reference domain file. See Usage Examples|
Perform exhaustive search against HMM database (default: heuristic search)
Output best non-overlapped domain hits of the AS gene. (Default: output best non-overlapped domain hits of each variant).
Output all different overlapped domain hits of each alternative variant.(Default: output the best non-overlapped domain hits of each variant).
|[-l|--local]||Run local search mode. (Default: glocal)|
|[(-o|--overlapping) <OVERLAPPING>]||Hit overlapping threshold (integer) (default: 0)|
Print on standard output the sequences of reference transcript of each gene on FASTA format. This parameter is only used with--gtf and --genome parameters.
Performs a domain search only on constitutive regions of all genes (Method to obtain results for the paper)
Run Näive search. Search domains against all genes with alternative splicing without AstaFunk Heuristics. Needs a reference domain file (Method to obtain results for the paper).
Search HMM database (--hmm) against FASTA sequences. (Method to obtain results for the paper).
Path to FASTA Sequence file. This file is used as input to evaluate the method employed by AstaFunk to align sequences (--test).
|[--cpu <CPU>]||Number of threads (Default: 1)|
In this section, we describe the optional and mandatory input data required to run AstaFunk:
<HMM_FILE> is an unique profile HMM or multiples HMMs in the same file (with extension .hmm) of the Pfam-A database from Pfam. You can download the complete Pfam-A database from FTP site: ftp://ftp.ebi.ac.uk/pub/databases/Pfam/current_release/Pfam-A.hmm.gz or download individual profiles using the family browser: http://pfam.xfam.org/family/browse.
<GTF> is the gene annotation based on GTF (Gene Transfer Format) format file of the input genome.
<GENOME_DIR> is the directory path to FASTA files (one chromosome per file) of the genome assembly.
Assume your annotation GTF file is (some fields are hidden after coordinates):
So, the FASTA files in the directory <GENOME_DIR> must be chr1.fasta, chr2.fasta, chr3.fasta, chr4.fasta and chr5.fasta.
Reference domain file with predicted domains for the reference transcript of each alternatively spliced gene. See how to create a reference domain file on Usage Examples.
AstaFunk prints on standard output the predictions of domains for each variant. See below column names of the standard output (tab-separated):
Code or pattern and splice chain (pipe-separated) of each AS event overlapped by the domain hit. The events are single-whitespace separated. Example:
To learn more about AS event patterns see references on 3.1 - Tool ASTA (AS Event Retriever).
The parameters without brackets are mandatory for the respective mode. Otherwise, it is optional. Parameters between pipe ("|") are mutually exclusive.
Observation: On AS genes, the current version of this mode searches constitutive domains only on reference transcript (longest ORF).
Searches exhaustively the HMM database against the variant sequences, i.e., without a reference domain file.
You can view the complete javadoc of barna on http://sammeth.net/jenkins/job/barna-devel/javadoc/: AstaFunk documentation can be found on packages barna.astafunk.*