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Introduction

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.

Obtaining AstaFunk

source code

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.

$> git clone http://bitbucket.sammeth.net/bitbucket/scm/barna/barna.git
Cloning into 'barna'...
remote: Counting objects: 29522, done.
remote: Compressing objects: 100% (11638/11638), done.
remote: Total 29522 (delta 11254), reused 27997 (delta 10681)
Receiving objects: 100% (29522/29522), 99.43 MiB | 706.00 KiB/s, done.
Resolving deltas: 100% (11254/11254), done.
Checking connectivity... done.
 
$> git checkout vitorafunk_dev_fix1stable
Branch vitorafunk_dev_fix1stable set up to track remote branch vitorastafunk_dev_fix1stable from origin.
Switched to a new branch 'vitorafunk_dev_fix1stable'

Building binaries

Build the binaries of AStalavista and create a distribution version.

$> cd barna/
$> cd barna.astalavista/
$> ../gradlew dist
.
. {some log messages}
.
BUILD SUCCESSFUL
Total time: 2 mins 11.574 secs

Extracting binary files

Enter into the distribution directory and extract the files (.tgz or .zip). In barna.astalavista directory:

$> cd build/distributions/
$> unzip astalavista-34.20.1-SNAPSHOT.zip

Check build

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

$> ./astalavista -t astafunk --help

You You will see:

$> ./astalavista -t astafunk --help
[INFO] Astalavista v4.0 (Flux Library: 1.30)

-------Documentation & Issue Tracker-------
Barna Wiki (Docs): http://confluence.sammeth.net/confluence
Barna JIRA (Bugs): http://jira.sammeth.net/jira

Please feel free to create an account in the public
JIRA and reports any bugs or feature requests.
-------------------------------------------

Current tool: astafunk
Search HMM-profiles of protein families (Pfam) on alternatively spliced genes.
Tool specific options:
.
. {help messages}
.

Input data

In this section, we describe the optional and mandatory input data required to run AstaFunk:

--hmm <HMM_FILE.hmm>

<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 <GTF_FILE.gtf>

<GTF> is the gene annotation based on GTF (Gene Transfer Format) format file of the input genome.

Image Removed If you only have a GFF annotation file, convert to GTF using gffread of Cufflinks or other script.

--genome <GENOME_DIR>

<GENOME_DIR> is the directory path to FASTA files (one chromosome per file) of the genome assembly.

chr1 hg19_refGene start_codon  67000042    67000044 ...;
chr2 hg19_refGene start_codon  201173796   201173798  ...;
chr3 hg19_refGene exon         134204575   134204894 ...;
chr4 hg19_refGene start_codon  41937502    41937504 ...;
chr5 hg19_refGene start_codon  134210118   134210120 ...;

-r|--reference <REFERENCE_FILE>

Reference file with predicted domains for the reference transcript of each alternatively spliced gene.

Image Removed How to Create a Reference File

<REFERENCE_FILE> is computed by hmmsearch of the HMMER program, using the command line below:

$> hmmsearch --cut_ga --domtblout <REFERENCE_FILE> Pfam-A.hmm <REFERENCE_TRANSCRIPTS.fasta>

hmmsearch is the HMMER algorithm (hmmer.org) to search one or more profiles (from the Pfam-A.hmm database) against the amino acid sequences of reference transcripts (in the <REFERENCE_TRANSCRIPTS>.fasta, see help below). The parameter --cut_ga is that hmmsearch uses gathering domain thresholds stored in the HMM profiles during predictions. The --domtblout output saves a parseable table of per-domain hits to <REFERENCE_FILE>. The reference transcript is the transcript with the longest ORF of a gene.

Image Removed Using AstaFunk to Generate a Multi-fasta File with the Reference Transcripts

AstaFunk includes a feature to generate a multi-fasta file with the amino acid sequences of reference transcripts for a given annotation.

Firstly, you execute ASTAFUNK to print on standard output (redirected to the file <REFERENCE_TRANSCRIPTS.fasta>) the amino acid sequences of the reference transcripts. A reference transcript is the transcript with the longest Open Reading Frame (ORF) of an alternatively spliced gene.

Obtain the reference transcript FASTA file with the command:

$> astalavista -t astafunk --tref --genome <GENOME_DIR> --gtf <GTF_FILE.gtf> > <REFERENCE_TRANSCRIPTS.fasta>

Lastest Release

Options

Input data

In this section, we describe the optional and mandatory input data required to run AstaFunk:

--hmm <HMM_FILE.hmm>

<HMM_FILE> is an unique profile HMM or multiples HMMs in the same file (with extension .hmm) of the Pfam-A database. You can either download the complete Pfam-A database from the EBI FTP site or specific Pfam profiles using the Pfam family browser.

--gtf <GTF_FILE.gtf>

<GTF> is the gene annotation based on GTF (Gene Transfer Format) format file of the input genome.

Image Added If you only have a GFF annotation file, convert to GTF using gffread of Cufflinks or other script.

~$ cat ce6_original.gtf | awk -v FS="\t" -v OFS="\t" '{if($3=="CDS") {print $0; $3="exon"; print $0}}' > ce6.gtf

--genome <GENOME_DIR>

<GENOME_DIR> is the directory path to FASTA files (one chromosome per file) of the genome assembly.

chr1 hg19_refGene start_codon  67000042    67000044 ...;
chr2 hg19_refGene start_codon  201173796   201173798  ...;
chr3 hg19_refGene exon         134204575   134204894 ...;
chr4 hg19_refGene start_codon  41937502    41937504 ...;
chr5 hg19_refGene start_codon  134210118   134210120 ...;

-r|--reference <REFERENCE_FILE>

Reference domain file with predicted domains for the reference transcript of each alternatively spliced gene. See below how to create a reference domain file.

Reference Domain file

<REFERENCE_FILE> is computed by hmmsearch of the HMMER program, using the command line below:

$> hmmsearch --cut_ga --domtblout <REFERENCE_FILE> <HMM_FILE> <REFERENCE_TRANSCRIPTS.fasta>

hmmsearch is the HMMER algorithm (hmmer.org) to search one or more profiles (from the Pfam-A.hmm database) against the amino acid sequences of reference transcripts (in the <REFERENCE_TRANSCRIPTS>.fasta, see help below). The parameter --cut_ga is that hmmsearch uses gathering domain thresholds stored in the HMM profiles during predictions. The --domtblout output saves a parseable table of per-domain hits to <REFERENCE_FILE>. The reference transcript is the transcript with the longest ORF of a gene. See below how to obtain the reference transcript FASTA file <REFERENCE_TRANSCRIPTS.fasta>.

Reference transcript sequences <REFERENCE_TRANSCRIPTS.fasta>

AstaFunk includes a feature to generate a multi-fasta file with the amino acid sequences of reference transcripts for a given annotation. Firstly, you execute ASTAFUNK to print on standard output (redirected to the file <REFERENCE_TRANSCRIPTS.fasta>) the amino acid sequences of the reference transcripts. A reference transcript is the transcript with the longest Open Reading Frame (ORF) of an alternatively spliced gene.

$> astalavista -t astafunk --tref --genome <GENOME_DIR> --gtf <GTF_FILE.gtf> > <REFERENCE_TRANSCRIPTS.fasta>

Program Output

AstaFunk

Program Usage

The basic search command of AstaFunk is:

$> astalavista -t astafunk --gtf <GTF_FILE.gtf> --genome <GENOME_DIR> --hmm <HMM_FILE.hmm> --reference <REFERENCE_FILE>

Options

Program Output

AstaFunk prints on standard output the predictions of domains for each variant. See below column names of the standard output (tab-separated):

1. chr: Field "seqname" of the GTF annotation; name of the chromosome or scaffold; Example: “chr1”.

2. loci_cluster

: string of concatenated AS transcript/gene identifiers clustered in the same loci. Example:

“uc001dhm.2,uc001dhn.3,uc001dho.3”.

3

.

domain_

cluster: a loci cluster has one or more domain clusters. Domain clusters are predictions of the same domain that overlap by genomic coordinates. It is integer number. In the cartoon below, domains "Blue" and "Yellow" were predicted on 3 variants of a loci. Predictions A, B and C are predictions of the same domain and they are overlapping by their genomic coordinates, resulting in the Cluster 1. Prediction D of domain "Blue" is overlapping the prediction F, but prediction F is from another domain "Yellow". It results in the Cluster 2. Finally, predictions E and F are from domain "Yellow" and they are overlapping the same genomic region, forming the Cluster 3.

Image Added4. merged_variant: set of transcripts with same exon/intron composition between the first source and last sink. If variants contains a list of transcripts identifiers separated by commas means that the transcripts have the same exon/intron composition between the first source and last sink.

Getting Started

Searching protein domains on alternatively spliced regions of human gene TNNT1

According to RefSeq (NM_003283), 

This gene encodes a protein that is a subunit of troponin, which is a regulatory complex located on the thin filament of the sarcomere. This complex regulates striated muscle contraction in response to fluctuations in intracellular calcium concentration.

Input

Command line

$> astalavista -t astafunk --tref --gtf tnnt1.gtf --genome ~/example/genome/ > reference_tx.fasta

$> hmmsearch --domtblout reference_file ~/Databases/Pfam/Pfam-A.hmm reference_tx.fasta

$> grep -v "#" reference_file | awk '{print $5}' | sort | uniq > list-hmm-tnnt1
$> hmmfetch -f Pfam-A.hmm list-hmm-tnnt1 > database.hmm

astalavista -t astafunk --genome ~/example/genome/ --gtf tnnt1.gtf --reference reference_file --hmm database.hmm

Extra: Constitutive Domains

astalavista -t astafunk --const --genome ~/example/genome/ --gtf tnnt1.gtf --reference reference_file --hmm database.hmm

Output

AstaFunk identifies six complete alternative events between the eight alternative transcripts of the gene TNNT1 (in the paper we present, just for an example, only two events). See the standard output:

When different variants resulting in predictions with same domain, score, and genomic coordinates, these predictions are collapsed in a unique prediction. In this case, the variants collapsed are separated by semi-colons (";");

5. acc: Accession number of the profile HMM. Example: “PF00406.19”. Other possible values for this field:

• <ACC_NUMBER>-NO_CDS: AStalavista in silico translation was not performed because the transcript does not have a valid CDS: CDS sequence is not multiple of three or it has a in-frame stop codon.
• <ACC_NUMBER>-NO_HIT: it was expected a domain <ACC_NUMBER>, but it was not predicted in the alternative splicing event region.

6. bitscore: Bit score of the alignment.

7. start_seq: Start position of the alignment in the sequence. This field can be a semi-colon-separated list of start positions, indicating that another prediction with the same domain, score and genomic coordinates was collapsed in this prediction.

8. end_seq: End position of the alignment in the sequence. This field can be a semi-colon-separated list of end positions, indicating that another prediction with the same domain, score and genomic coordinates was collapsed in this prediction.

9. start_genomic : Start position of the alignment in the genome

10. end_genomic: End position of the alignment in the genome

11. first_source : Source is the start genomic position of the fused AS events.

12. last_sink: Sink is the end genomic position of the fused AS events.

13. start_model: Alignment start state of the profile HMM.

14. end_model: Alignment end state of the profile HMM

15. length_model: number of states of the profile HMM

16. event code: code of events overlapped by the domain prediction. The pipes "|" separate the codes of multiple events. This field can be a semi-colon-separated list, indicating that another prediction with the same domain, score and genomic coordinates was collapsed in this prediction.

17. splice chain: splice chain of events overlapped by the domain prediction. The pipes "|" separate the splice chains of multiple events. This field can be a semi-colon-separated list, indicating that another prediction with the same domain, score and genomic coordinates was collapsed in this prediction.

18. variant list: variant list of events overlapped by the domain prediction. Different variants of the same event are enclosed by brackets, e.g., [tx₁][tx₂, tx₃] are two variant of a event e where the 1^st variant is represented by tx₁ and the 2^nd variant is represented by tx₂ and tx₃. This field can be a semi-colon-separated list, indicating that another prediction with the same domain, score and genomic coordinates was collapsed in this prediction. The pipes "|" separate the variant list of multiple events:

To learn more about AS event patterns see references on 3.1 - Tool ASTA (AS Event Retriever).

Mode Summary

The parameters without brackets are mandatory for the respective mode. Otherwise, it is optional. Parameters between pipe ("|") are mutually exclusive.

Search Alternatively Spliced Domains

Search Pfam protein domains on alternatively spliced regions of the variants. This mode uses a series of heuristics to avoid redundant scans on AS-enriched transcriptomes.

astalavista -t astafunk [--verbose] [--cpu <INT>] [--all | -g] [--local] [-o <INT>] --genome <GENOME_DIR> --gtf <GTF_FILE> --hmm <HMM_FILE> --reference|-r <REFERENCE_FILE>

The parameter --all will report all prediction of different domains (even they are overlapping in genomic coordinates) of each variant of an AS gene. The parameter -g will report the best domain hits (non-overlapping in genomic coordinates) among all variants of a gene, i. e. it will report the best non-overlapped domain predictions of an AS gene. The default reporting is best non-overlapped domain prediction of each variant of an AS gene.

Search Constitutive Domains

Search constitutive domains, i. e. report the domain hits found on constitutive exons. On AS genes, AstaFunk searches domains only on the sequence of the reference transcript, i. e. the transcript with the longest ORF. Otherwhise, AstaFunk reports all domain hits of the non-AS genes.

astalavista -t astafunk  [--verbose] [--cpu <INT>] [--local] [-o <INT>] --const --genome <GENOME_DIR> --gtf <GTF_FILE> --hmm <HMM_FILE> --reference|-r <REFERENCE_FILE>

Search AS domains exhaustively

Searches exhaustively, i. e.,  without a reference domain file, the HMM database against the variant sequences.

astalavista -t astafunk  [--verbose] [--cpu <INT>] [--all | -g] [--local] [-o <INT>] -e|--exh --genome <GENOME_DIR> --gtf <GTF_FILE> --hmm <HMM_FILE>

Näive Search

Search the HMM database against each transcript of an AS gene. Differently from "Search Alternatively Spliced Domains" mode, this mode searches the entire sequence of all transcripts, potentially resulting in redundant searches on the AS-enriched transcriptomes and constitutive regions of the transcripts.

astalavista -t astafunk  [--verbose] [--cpu <INT>] [--local] [-o <INT>] --naive --genome <GENOME_DIR> --gtf <GTF_FILE> --hmm <HMM_FILE> --reference|-r <REFERENCE_FILE>

Print the reference transcript sequences

Print on standard output the FASTA sequences of (reference) transcripts of (AS-)genes on <GTF_FILE>.

astalavista -t astafunk  --tref --genome <GENOME_DIR> --gtf <GTF_FILE>

Search HMM database against FASTA sequences

Mode to search a HMM database against a FASTA sequence file.

astalavista -t astafunk [--local] [-o <INT>]--test --hmm <HMM_FILE> --fa <SEQUENCE_FILE>

Documentation of the JAVA source code