README for TEMT (1.0)

* Introduction

The next-generation sequencing technology RNA-Seq keeps growing as
a major method for transcriptome research, e.g. transcript abundances
estimation. However, the tissue heterogeneity problem widely presented
in native tissue environment, can substantially confound the transcript
abundances estimation of each individual cell type. Although experimental
methods have been proposed to dissect multiple distinct cell types,
computationally ``denoising'' heterogeneous tissue also stands out as a
reliable alternative due to its own advantages, such as keeping the tissue
sample as well as the subsequent molecular content yield intact. Here we
propose a probabilistic model-based approach, Transcript Estimation from
Mixed Tissue samples (TEMT), to estimate the transcript abundances of
single cell type of interest via the RNA-Seq data from heterogeneous
tissue sample. TEMT has incorporated the positional and sequence-specific
bias, and with the online EM algorithm adopted, it has a time requirement
proportional to the data size and a constant memory requirement. We tested
the proposed method on a series of datasets from both simulation and ENCODE
data, and the results of TEMT outperforms current directly estimation method.

* Prerequisite

Python version must be equal to 2.6 or 2.7 to run TEMT. We recommend
using the version 2.6.5.

numpy-1.3.0 or higher is required. numpy distributions can be found at:
http://www.numpy.org/
http://sourceforge.net/projects/numpy/files/

scipy-0.7.0 or higher is required. scipy distributions can be found at:
http://www.scipy.org/
http://sourceforge.net/projects/scipy/files/

pygr-0.8.2 is required. pygr distributions can be found at:
http://code.google.com/p/pygr/

* Usage

Usage: TEMT.py [options] <-p pfile> <-m mfile> <-t tfile>  

Example: TEMT.py -p pure.sam -m mix.sam -t transcripts.fasta -P 0.9 -l 75 -a type_a -b type_b -A 0 --bias-module 


Options:
  --version             show program's version number and exit
  -h, --help            show this help message and exit
  -p INREADS_P, --pure-sample=INREADS_P
                        read alignment file of the pure sample in SAM format.
                        REQUIRED
  -m INREADS_M, --mixed-sample=INREADS_M
                        read alignment file of the mixed sample in SAM format.
                        REQUIRED
  -t INTRANSCRIPTS, --transcripts=INTRANSCRIPTS
                        reference transcripts file in fasta format. REQUIRED
  -a OUTREADS_A, --type-a=OUTREADS_A
                        the name of the output file of cell type a, which is
                        the only cell type of the pure sample. DEFAULT:
                        "type_a"
  -b OUTREADS_B, --type-b=OUTREADS_B
                        the name of the output file of cell type b, which is
                        the second cell type within the mixed sample. DEFAULT:
                        "type_b"
  -P B_PROP, --type-b-proportion=B_PROP
                        cell type b proportion. e.g. "-P 0.9". REQUIRED
  -l READ_LEN, --read-length=READ_LEN
                        read length. REQUIRED
  -A ADD_ROUNDS, --additional-rounds=ADD_ROUNDS
                        the number of addtional rounds of EM algorithm after
                        the first online round. DEFAULT: 0
  --bias-module         enable the positional and sequence specific bias
                        module. DEFAULT: False
				
				
* Example

In the example_data directory, we presents two pairs of 75-bp read sets under directories
b_proportion_50 and b_proportion_90 respectively, and one transcript set transcripts.fasta.
For each pair of read sets, one is from pure tissue of cell type a, named type_a.fastq;
while the other is from mixed tissue of cell type a and b, mixed with type b proportion,
named mix.fastq.

To run TEMT, first we need to align the read sets to the transcript set transcripts.fasta.
We recommend to use bowtie for the reads alignment. e.g.

bowtie-build transcripts.fasta transcripts
bowtie -n 2 -k 10 -S ../transcripts type_a.fastq > type_a.sam
bowtie -n 2 -k 10 -S ../transcripts mix.fastq > mix.sam

Then, we can run TEMT with 1 round online EM and bias-module disabled under b_proportion_90 directory:

TEMT.py -p type_a.sam -m mix.sam -t ../transcripts.fasta -P 0.9 -l 75 -a type_a -b type_b

We can also run TEMT with additional online EM rounds and bias-model enabled, which both of
these two options improve the accuracy:

TEMT.py -p type_a.sam -m mix.sam -t ../transcripts.fasta -P 0.9 -l 75 -a type_a -b type_b -A 1 --bias-module

* Output files

1. type_a.temt is the relative transcript abundances of cell type a in terms of estimated counts and RPKM.
2. type_b.temt is the relative transcript abundances of cell type b in terms of estimated counts and RPKM.

Estimated counts is the esimated number of reads from the read set that originated from the target transcript.

RPKM represents Reads Per Kilobase per Million mapped reads, which is a method of quantifying transcripts 
expression from RNA-Seq data by normalizing for total transcript length and the number of sequencing reads.


* Reference
This work is mainly based on:
(Li and Xie, BMC Bioinformatics, 2013). 
The default parameter settings and some of the features are using:
(Roberts and Pachter, Nature Methods, 2012)
http://bio.math.berkeley.edu/eXpress/overview.html