#NLDMseq

#What is NLDMseq?

NLDMseq is the software for expression calculation at both gene and isoform levels from RNA-seq data given a reference transcriptome. The program calculates expression values using the alignment from Bowtie 2.

The software is an open-source software. You can redistribute it and/or modify it under the terms of the GNU General Public License (GPL) as published by the Free Software Foundation.

#How NLDMseq works?

##Features

• NLDMseq adopts latent variables to model the unknown isoforms, from which reads originate, and the underlying percentage of multiple spliced variants.

• NLDMseq provides an approach to accurately estimate gene and isoform expression from RNA-Seq data by modeling the isoform- and exon-specific read sequencing biases.
• NLDMseq provides the measurement uncertainty associated with the estimated gene and isoform expression.

#Contact Users can ask technical questions by sending emails to Xuejun Liu (xuejun.liu@nuaa.edu.cn).

#Installation

##Installation requirements:

• Operating system : Linux( Ubuntu13.10 or Fedora 17…) Mac OS X (Mac OS X 10.8 5 or higher)
• Python 2.7
• GUN Scientific Library GCC 4.8.1 or higher(http://www.gnu.org/software/gsl/)
• Python package: numpy,parallel python (pp)(http://www.parallelpython.com/)
• Bowtie2

###NOTE: PP is a python module which provides mechanism for parallel execution of python code on SMP(systems with multiple processors or cores) and clusters (computers connected via network). It is light, easy to install and integrate with other python software. PP is an open-source and cross-platform module written in pure python.

The software depends on the free and open-source software, the GNU Scientific Library (GSL) (http://www.gnu.org/software/gsl/), so the GSL needs to be installed on the user’s system. NLDMseq requires the GSL installed in /usr/local, which is the default location. The GSL can be compiled by the user. Users who are compiling NLDMseq from the source code should install GSL in the standard location (/usr/local). If you are not sure if GSL is already installed, at the Terminal prompt $ type:$gsl-config --cflags --libs-without-cblas If GSL is installed, the command above should return the following information:

-I/sw/include
-L/sw/lib -lgsl –lm

GSL can be found in the gsl subdirectory on your nearest GNU mirror ( http://ftpmirror.gnu.org/gsl/) or the Main GNU ftp site (ftp://ftp.gnu.org/gnu/gsl/). The users can download gsl-1.6.tar.gz or a higher version for NLDMseq. Please follow the instructions in the included file “INSTALL” to guide the installation of this library.

We recommend using the Linux operating system. The test shows that it will cost much more time on a Mac OS X machine.

##Installation

• First, go to the location of NLDMseq

    $cd ~

• Second, install the software by the install.sh shell script.

$./INSTALL.sh

NOTE:The user may have to give the script execute permissions by chmod u+x INSTALL.sh. This script will use the gcc complier to build C codes in the software.

• Test the software

$python test_NLDMseq

To test this software, the user can run the python script test_NLDMseq.py .This will check whether the software has been built and installed correctly. It will calculate the expression of genes and isoforms using the data from the TEST_DATA folder.

##Running NLDMseq

• Step 1. Aligning sequenced reads with Bowtie 2 The following commands are used to align sequenced reads to a reference transcriptome.

$ bowtie2-build -f ref_transcript. Fasta ref_transcript.index
$ bowtie2 –t –f -k 20 -p 4--no-sq --no-head -x ref_transcript.index raw_data.fasta -S align_reads.output

If the paired-end reads are processed, the Bowtie command should be listed as

$ bowtie2 –t–f -k 20 -p 4 --no-sq --no-head -x ensGene.ref_transcript.index -1 raw_data.fasta -2 raw_data.fasta -S align_reads.output

Note:

NLDMseq is designed for SAM file ,so -S parameter is necessary. The above transcriptome reference sequence can be downloaded from UCSC or Ensembl website.

• Step 2. Calculate expression values with NLDMseq

The software uses the alignment SAM file from Bowtie 2 as input data.

$ python NLDMseq-calulation.py –a AnnotationFile.txt  -t AnnotationFile Type –i align_file(s) –o  expression_path

Options:

• -a/--AnnotationFile: The annotation includes gene and isoform information. (e.g: refGene, knownGene, and ensGene, which can all be downloaded from UCSC website.)
• -t/--AnnotationType : an integer to select the type of annotation. refGene:1, ensGen:2, knownGene:3 and Ensmbl: 4.
• -i/--Input: Alignment file(s), which is Bowtie 2 output. If more than one output files for multiple lanes under a condition are considered, use comma for the separation(e.g. align_reads1.output, align_reads2.ouput).
• -o/--OutputPath All output files and temp files during the process of calculation are produced under this given path. (E.g. two output files will be produced, named expression_gene.txt and expression_isofrom.txt)
• -s/--Single-end: Optional. Input data are alignment for single-end reads. (Default: off)
• -h/-Help: Show help information.

Output files: The NLDMseq produces gene and isoform expression output files, which are put under the path given in the option -o/--OutputPaht

Table 1: Description of output files:

Column Nr	Name	Description
1	gene/isoform Name	Name of a gen/Name of gene and isoform
2	FPKM	FPKM expression value of gene/isoform.
3	Variance of FPKM	The variance value of estimated gene/isoforme expression

#Example Here, we use a simple example to show the usage of NLDMseq. The alignment files from Bowtie 2 and the annotation file are also supplied.

• Annotation file: ensGene.txt
• Alignment from Bowtie2: SE_read_example.sam (single-end)
• Alignment from Bowtie2:PE_read_example.sam (paired-end)

Since the Bowtie2 output has been supplied, so you can skip step1 and just run the following command.

For single-end data:

$ python NLDMseq-calulation.py –a ../example_data/annocation/annocation.gtf  -t 4 -i ../example_data/SE_example –o ../se_out -s 

After running the above command, you will obtain two output files of this single-end data, expression_gene.txt and expression_isoform.txt under se_out path.

For paired-end data :

$ python NLDMseq-calulation.py –a ../example_data/annocation.gtf –t 4 –i ../example_data/PE_example –o../pe_out

After running the above command, you will obtain two output files of this paired-end data, expression_gene.txt and expression_isoform.txt under pe_out path.

#Authors Xuejun Liu (xuejun.liu@nuaa.edu.cn).College of Computer Science and Technology, Nanjing University of Aeronautics and Astronautics, 29Jiangjun Rd., Jiangning District, 211106Nanjing China. We use the C source code from Daichi Mochihashi (ATR Spoken Language Communication Research Laboratories, Kyoto, Japan daichi.mochihashi@atr.jp) #Related software

Bowtie2: for alignment of RNA-Seq reads to transcriptome (optionally with the precomputed set of splice junctions).