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# AmpliconArchitect
## AmpliconArchitect (AA) is a tool to identify one or more connected genomic regions which have simultaneous copy number amplification and elucidates the architecture of the amplicon.
## In the current version, AA takes as input next generation sequencing reads (paired-end Illumina reads) mapped to the hg19/GrCH37 reference sequence and one or more regions of interest.
## This is development version. Please "watch" this repository for announcement of production release and methods manuscript soon.
## Note: see chromosome name format for bamfile: 'chr1' .. 'chr22', 'chrX', 'chrY' as in "data_repo/hg19full.fa". (Thanks to Sitan Qiao, BGI for noticing this missing information).

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# Installation:

# AA download (if you have not already cloned this source code):
git clone https://github.com/virajbdeshpande/AmpliconArchitect.git

# Dependencies:
## 1) Python 2.7

## 2) Ubuntu libraries and tools:
sudo apt-get install build-essential python-dev gfortran python-numpy python-scipy python-matplotlib python-pip zlib1g-dev samtools 

## 3) Pysam verion 0.9.0 (https://github.com/pysam-developers/pysam):
sudo pip install pysam

## 4) Mosek optimization tool (https://www.mosek.com/):
wget http://download.mosek.com/stable/7.1.0.12/mosektoolslinux64x86.tar.bz2
tar xf mosektoolslinux64x86.tar.bz2
echo Please obtain license from https://mosek.com/resources/academic-license or https://mosek.com/resources/trial-license and place in $PWD/mosek/7/licenses
echo export MOSEKPLATFORM=linux64x86 >> ~/.bashrc
export MOSEKPLATFORM=linux64x86
echo export PATH=$PATH:$PWD/mosek/7/tools/platform/$MOSEKPLATFORM/bin >> ~/.bashrc
echo export LD_LIBRARY_PATH=$LD_LIBRARY_PATH:$PWD/mosek/7/tools/platform/$MOSEKPLATFORM/bin >> ~/.bashrc
echo export MOSEKLM_LICENSE_FILE=$PWD/mosek/7/licenses >> ~/.bashrc
cd $PWD/mosek/7/tools/platform/linux64x86/python/2/
sudo python setup.py install #(--user)
cd -
source ~/.bashrc


# Data repositories:
## Download the data repositories. While we include some annotations, we are unable to host some large files in the git repository.
## These may be downloaded from https://drive.google.com/open?id=0ByYcg0axX7udSjdZeUNqdlNVNHc . Thanks to Peter Ulz for noticing incorrect link earlier.
tar zxf data_repo.tar.gz
echo export AA_DATA_REPO=$PWD/data_repo >> ~/.bashrc
source ~/.bashrc

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# AmpliconArchitect reconstruction:

# Preprocessing:

## Input 1: Coordinate sorted bam file:
## Use bwa mem or any other software to align reads to the hg19full.fa in the data_repo human reference to output a bam file.
## Sort the bam file by genomic coordinates and index the coordinate sorted bam file using "samtools index".

## Input 2: Amplicon Interval(s):
## Identify interval(s) of interest by using a CNV caller e.g. ReadDepth, but may use others or user defined intervals.
## Recommended to use file AmpliconArchitect/src/read_depth_params for calling variants with ReadDepth.
## Recommended only analyze CN gains > 5x copy count and > 100kbp in size.
## Recommended to filter out intervals with significant overlap with conserved regions.
## Input intervals in bed file format
python src/amplified_intervals.py --bed {read_depth_folder}/output/alts.dat > {outName}.bed

# Amplicon reconstruction
python src/AmpliconArchitect.py --bam <input_bam> --bed <bed file> --out <prefix for output files>

# Output description
## The software generates 3 types of output files
## 1) <outName>_summary.txt:
## This file includes a summary for all amplicons detected
## 
## 2) <outName>_amplicon<id>_graph.txt:
## For each amplicon, breakpoint graph describing sequence edges and breakpoint edges in the graph
## Edge each consists of 2 vertices in the format <chromosome>:<position><orientation>
## e.g. A sequence edge chr1:10001-->chr1:20000+ represents the 10000bp genomic sequence. A breakpoint edge chr1:10001-->chr1:20000+ implies that on chr1, the forward strand from position 20000 is continues onto the forward strand at position 10001 in the 5' to 3' direction to create a 10000bp loop. Breakpoint vertices with position -1 are reserved for source vertex at the end of linear contig.
## 
## 3) <outName>_amplicon<id>_cycle.txt:
## For each amplicon, this file represents the list of structures contained in the amplicon.
## Segments represent genomic intervals (with possible overlaps) and each segment is assigned a unique id.
## Segment id 0 is reserved for source vertices. 
## Cycles are represented as ordered lists of connected segments along with orientation of the segments.
## The last segment connects back to the first segment of the cycle.
## Linear contigs have 0+ and 0- as their first and last segments.

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