CLASS - Constraint-based Local Assembly and Selection of Splice variants

Described in:

Current (dynamic-programming) version:

Song, L., Sabunciyan, S., and Florea, L. (2016). CLASS2: accurate and efficient splice variant annotation from RNA-seq reads. Nucleic Acids Research 2016; doi: 10.1093/nar/gkw158

Previous ('set cover') version:

Song, L. and Florea, L. (2013). CLASS: Constrained Transcript Assembly of RNA-seq Reads. Third Annual RECOMB Satellite Workshop on Massively Parallel Sequencing - RECOMB-SEQ 2013. BMC Bioinformatics 14(Suppl. 5), S14.

Copyright (C) 2012-2013, and GNU GPL, by Li Song, Liliana Florea

Includes portions copyright from:

lp_solve - Copyright (C) 2005, and GNU LGPL, by Michel Berkelaar, Kjell Eikland, Peter Notebaert

SAMtools - Copyright (C) 2008-2009, Genome Research Ltd, Heng Li

I. What is CLASS?

CLASS is a tool for assembling transcripts from short RNA-seq reads aligned to a reference genome. It works in three stages. Stage 1 makes use of linear programming to determine a set of exons. Stage 2 builds a splice graph representation of a gene, by connecting the exons (vertices) via introns (edges) extracted from spliced read alignments. Stage 3 selects a subset of the candidate transcripts encoded in the graph, according to the constraints derived from mate pairs and spliced alignments and, optionally, using knowledge about gene structure extracted from known annotation and/or alignments of cDNA sequences.

II. Install

Run: % sh build.sh

The software is compiled in place

III. Usage

Usage: perl run_class.pl [options]
Options:
	-a alignment_file (REQUIRED): the path to the alignemtn file(in SAM or BAM format)
	-o output_file: the file storing the output of CLASS (default: ./alignment_file_wo_extension.gtf)
	-p number_of_threads: specify the number of worker threads (default:1)
	-F f: do not report the transcripts whose abundance level is lower than f*|most expressed transcript| in a gene
	-l label: add a prefix and a "_" to the ids in the GTF file (default: not used)
	-j junction: the path to the splice junction file
	-e evidence: the path to the evidence files
	--var-rd-len: extensive variable read lengths, i.e. reads after trimming (default: auto-detect)
	--set-cover: use set cover to build transcripts from splicing graph (default: no)
	--verbose: also output the procedure of CLASS (default: no)
	--wd tempoaray_file_directory: the directory storing the temporary files (default: ./class_tmp)
	--clean: whether to remove the temporary files in -wd (default: no)

IV. Input/Output

The primary input to CLASS is a set of short read alignments in BAM format and sorted by chromosome and position, for instance one produced with the program Tophat2.

CLASS requires XS field in BAM file to know the strand of spliced alignment. If the tools does not provide XS field, such as the default behavior of STAR, you can use the program "addXS" in the package to add XS field to the BAM file. You can run "addXS" as:

./addXS reference_genome.fa < sam_file

The typical usage with BAM file can be:

samtools view -h in.bam | ./addXS reference_genome.fa | samtools view -bS - > out.bam 

Given an alignment input x.bam, CLASS produces two intermediate data files, x.depth and x.splice in the temporary working directory.

• The format of the x.depth file, generate by samtools, is: chrom_id position #_of_reads_on_the_position

• The format of the x.splice file, generate by 'junc', is:

  chrom_id start_intron_position end_intron_position #_of_supporting_reads strand #_unique_support_read #_multi-aligned_support_read sum_of_unique_support_reads_edit_distance sum_of_multi-aligned_support_reads_edit_distance

  NOTE: When using the '-a' argument, the value #_of_supporting_reads can be negative, indicating that this splice junction is invalid.

Lastly, to produce a set of transcripts, the program 'class' takes as input a BAM/SAM file, the depth file generated by 'samtools' and the splice junction file generated by 'junc'. The final output, consisting of predicted transcripts, is in standard GTF format.

NOTE: 'samtools' can only read a BAM file, while 'junc' and 'class' can read both SAM and BAM formats.

IMPORTANT: Please make sure to enable the appropriate access to the directory containing the temporary workind girectory. Also, since the depth file will be created in that directory and will have one row for each base, please ensure that sufficient space is available in the work directory.

V. Example

We will use the file ./Sample/sample.bam as an example. Running "% perl run_class.pl -a ./Sample/sample.bam" will produce a set of transcripts in the file ./sample.gtf .

VI. 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 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

VII. Support

Contact us at: [email protected], [email protected]