Segmentum a fast tool for copy number analysis of cancer genome
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README.md

Segmentum

Segmentum: a tool for copy number analysis of cancer genome.

Introduction

Segmentum is a tool for the identification of CNAs and copy-neutral loss of heterozygosity (LOH) in tumor samples using whole-genome sequencing data. Segmentum segments the genome by analyzing the read-depth and B-allele fraction profiles using a double sliding window method. It requires a matched normal sample to correct for biases and to discriminate somatic from germline events. Segmentum, written in the Python programming language, is fast and performs segmentation of a whole genome in less than two minutes.

Citation information

Scientific paper corresponding to this work is openly accessible online at BMC Bioinformatics.

Afyounian, E., Annala, M. and Nykter, M., 2017. Segmentum: a tool for copy number analysis of cancer genomes. BMC Bioinformatics, [online] 18(215), pp.1-10. Available at: https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-017-1626-8

Installation

To install Segmentum, download the latest release and extract it to a folder. Then run the Makefile and add the subfolder bin/ to your PATH. See below for an example:

wget https://github.com/eafyounian/Segmentum/archive/master.zip   
unzip master.zip   
cd Segmentum-master/    
make   
export PATH=/some/folder/Segmentum-master/bin:$PATH   

Segmentum requires Samtools to be installed.

Input files

1. Tumor and its matched normal coverage files with WIG format.

Example

    fixedStep chrom=chr20 start=1 step=1000
    140
    151
    159
    144
    131

2. B-allele-fraction file containing the B-allele fractions at heterozygous SNPs.

Example

CHROM   POSITION    REF     ALT     sample_x_t
chr1    10174       C       T       0.00
chr1    10177       A       C       0.08
chr1    10583       G       A       0.00
chr1    15211       T       G       0.71
chr1    16487       T       C       0.15

Output files

Segmentum outputs a SEG file containing the identified segments.

Example

'ID              chrom   loc.start       loc.end     num_probes      seg.mean        baf.mean
sample_x_t       chr1    8000            80000       72              0.246188        0.425905
sample_x_t       chr1    80000           173000      93              -0.007791       0.365433
sample_x_t       chr1    173000          270000      97              -0.708895       0.487773

Running Segmentum

Segmentum can be run in two modes with/without using B-allele fraction data with the following commands:

## with BAF
Segmentum analyze with BAF <tumor> <normal> <BAF_file> <window_size> <clogr_threshold> <BAF_threshold> [-m N] [-l N] [-b N] [-p N] [-B N]     

## without BAF
Segmentum analyze without BAF <tumor> <normal> <window_size> <clogr_threshold> [-m N] [-l N]  


Options:  
    -h --help             Show this screen
    -m --min_read=N       Minimum number of reads from the normal sample to calculate the coverage log ratio [default: 50]  
    -l --logr_merge=N     Log ratio segments merging threshold [default: 0.15]  
    -b --baf_merge=N      B-allele fraction segments merging threshold [default: 0.05] 
    -p --print=N          If true, prints the results to standard output, otherwise to a file with the same name as the sample name [default: True]    
    -B --BAF=N            If true, creates a .WIG file for heterozygous SNP allele fractions to be opened in IGV [default: True]    
    

Example with BAF
Segmentum analyze with BAF sample_x_t.wig.gz sample_x_n.wig.gz B_allele_fraction.tsv.gz 11 0.7 0.3

Segmentum outputs the results to standard output by default. Use -p False to create a SEG file with the same name as the tumor file.

Visualization of the results in IGV

In order to visualize the results in IGV, a new file should be created containing only first 6 fields of the output file.

Example
cut -f1,2,3,4,5,6 sample_x_t.seg > IGV_sample_x_t.seg

IGV_sample_x_t.seg is now ready to be loaded in IGV.

Extracting copy neutral LOH regions

In order to extract copy neutral LOH regions from the output(s) use the following command:

Segmentum find recurrent cnLOHs <seg_files>... [-c N] [-t N]  
    
Options:  
    -c --clogr_thresh=N   Coverage logratio must be below this threshold to call a copy neutral LOH region [default: 0.1]    
    -t --baf_thresh=N     B-allele fraction must be below this threshold to call a copy neutral LOH region [default: 0.15]   

Example
Segmentum find recurrent cnLOHs sample_x_t.seg
Segmentum find recurrent cnLOHs sample_x_t.seg sample_y_t.seg sample_z_t.seg #in case of more samples

Creating the input files from BAM files

In order to create the coverage files, use the following command:

Segmentum extract read depth <BAM_file> <window_size> [-q N]    
        
Options:   
    -q --quality=N        Minimum mapping quality [default: 10]   

Example

Segmentum extract read depth sample_x_t.bam 2000 | gzip -c > sample_x_t.wig.gz
Segmentum extract read depth sample_x_n.bam 2000 | gzip -c > sample_x_n.wig.gz

In order to create the B-allele-fraction file, use the following command:

Segmentum calculate BAF <genome_fasta> <SNP_position> <tumor> <normal> [--hetz=N:R] [-q N] [-r REGION]   
        
Options:  
    -r <region>           Restrict analysis to chromosomal region     
    -q --quality=N        Minimum mapping quality [default: 10]     
    --hetz=N:R            Minimum evidence for heterozygous [default: 4:0.3]    

Example

Segmentum calculate BAF hg19.fa hg19_1000g_2014oct_SNPs.tsv.gz sample_x_t.bam sample_x_n.bam --hetz=4:0.3 -q20 | gzip -c > B_allele_fraction.tsv.gz

Note on SNP postion file: You can either use the provided SNP postion file or make one of your own. If you are planning to use the provided one you can download it from here: http://compbio.uta.fi/segmentum/hg19_1000g_2014oct_SNPs.tsv.gz. If you are planning to make your own SNP position file, the file should have the following format:

    chr     position
    1       10177  
    1       10235  
    1       10352  
    1       10352  
    1       10505  

Samples You can find a set of sample coverage and BAF files for experimentation with Segmenum here: http://compbio.uta.fi/segmentum/sample_files.tar.gz.

simulated data You also can find a set of simulated coverage and BAF data as well as its corresponding ground truth for experimentation here: http://compbio.uta.fi/segmentum/simulated_data_example.tar.gz.