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Alubaster

Detection of novel Alu exonization events from RNA-seq data.

Described in:

Florea L, Payer L, Antonescu C, Yang G and Burns K. (2021) Detection of Alu exonization events in human frontal cortex from RNA-seq data, Submitted.

Supplementary data described in the article can be found here.

Copyright (C) 2018-2021 and GNU GPL v3.0 by Liliana Florea, Lindsay Payer

Synopsis

Alubaster identifies candidate gene loci by locating mapped reads (‘anchors’) whose Alu-containing mates could not be found in the genome. It then applies two types of filters to select a more accurate subset of loci. The first, a signal filter, identifies read evidence for an Alu exonization event, by searching the mate’s sequence against a concatenation of the neighboring exons’ and Alu sequences while concomitantly ruling out false positive matches that could have resulted from local or more distant Alu elements in the genome. The second, a context filter, evaluates the likelihood of an event based on the strength of the signal versus the local context, in particular repeat content and proportion of signal-to-‘context’ matches.

Installation

Alubaster is written in Perl and shell. To download the source code, clone the current GitHub repository:

git clone https://github.com/splicebox/Alubaster.git

Prerequisites

Required bioinformatics packages: sim4db, oases, tophat2 and kraken. Follow the instructions for each program to install and compile, then update the paths in the file 'ALUBASTER.config.sh'.

Before you start

Before you start your first Alubaster run, you will need to prepare several reference data files:

  1. Download the GENCODE gene annotation file (version of your choice) from gencodegenes.org (GTF file). Save this file in a directory of your choice.
  2. Generate a header file for the genome fasta file, ideally in the same directory as the genome:
grep "^>" genome.fa > genome.fa.hdrs

Next, perform the following steps in the reference directory:

  1. Generate the annotation index:
 perl make_annot_index.pl  < gencode.vXX.annotation.gtf > gencode.vXX.annotation.Txpt2Gene
  1. Generate the exons file, for instance by converting the GTF annotation file to multi-block BED, filtering out single exon transcripts, and then extracting the individual exons in a BED file:
gtf2bed gencode.vXX.annotation.gtf  |  awk '{ if ($10!=1) print $_; }' | bedtools bed12tobed6 > gencode.vXX.mx.singl_exon.bed

or you can use your own scripts. The format for the output file is:

# chrom    from    to   enstranscriptID     0      strand
chr1    11868   12227   ENST00000456328.2       0       +
  1. You should find a copy of the kraken Alu sequence database in the ‘reference’ directory. If not already there, for instance because of file size restrictions, you can download a copy here. Copy this in the 'reference' directory, or elsewhere, and extract the files, for instance with 'gunzip' and 'tar -xvf'.
  2. Update the paths in the 'ALUBASTER.config.sh' file to point to your local files and software.

Usage

  runAlubaster.sh <SampleName> <TophatDir> <FastqDir> <OutputDir>

  Required parameters:
  
  <SampleName>  Sample name as it apears in fastq files (i.e., sample name would be ABC if fastq files are ABC_{1,2}.fastq.gz)
  <TophatDir>   Path to a directory containing tophat output ('accepted_hits.bam' and 'unmapped.bam') for this sample
  <FastqDir>    Path to fastq files directory
  <OutputDir>   Path to a directory where the analysis is done.  If it does not exist, it will be created.
                For each sample, a subdirectory will be created and all the out files for this run will be written there;
                e.g., for sample ABC the results will be written to <OutputDir>/ABC/

Terms of use

This program is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2 of the License, or (at your option) any later version.

This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.

You should have received (LICENSE.txt) a copy of the GNU General Public License v3.0 along with this program; if not, you can obtain one from http://www.gnu.org/licenses/gpl.txt or by writing to the Free Software Foundation, Inc., 59 Temple Place, Suite 330, Boston, MA 02111-1307 USA

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