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:
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 file with predicted domains for the reference transcript of each alternatively spliced gene.
AstaFunk prints on standard output the predictions of domains for each variant. See below column names of the standard output (tab-separated):
chr: Field "seqname" of the GTF annotation; name of the chromosome or scaffold; Example: “chr1”.
gene_cluster: string of concatenated AS transcript/gene identifiers. Example: “uc001dhm.2,uc001dhn.3,uc001dho.3”.
acc: Accession number of the profile HMM. Example: “PF00406.19”.
bitscore: Bit score of the alignment
start_seq: Startposition of the alignment in the sequence.
end_seq: End position of the alignment in the sequence.
start_genomic : Start position of the alignment in the genome
end_genomic: End position of the alignment in the genome
first_source : Source is the start genomic position of the fused AS events.
last_sink: Sink is the end genomic position of the fused AS events.
start_model: Alignment start state of the profile HMM.
end_model: Alignment end state of the profile HMM
length_model: number of states of the profile HMM
Contains the code and splice chain (pipe-separated) of each AS event overlapped by the domain hit. The events are single-whitespace separated. Example:
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.*