Pseudo-cgh of next-generation sequencing data
Project description
Overview
Next-generation sequencing of tumor/normal pairs provides a good opportunity to examine large-scale copy number variation in the tumor relative to the normal sample. In practice, this concept seems to extend even to exome-capture sequencing of pairs of tumor and normal. This library consists of a single script, ngCGH, that computes a pseudo-CGH using simple coverage counting on the tumor relative to the normal.
I have chosen to use a fixed number of reads in the normal sample as the “windowing” approach. This has the advantage of producing copy number estimates that should have similar variance at each location. The algorithm will adaptively deal with inhomogeneities across the genome such as those associated with exome-capture technologies (to the extent that the capture was similar in both tumor and normal). The disadvantage is that the pseudo-probes will be at different locations for every “normal control” sample.
Installation
There are several possible ways to install ngCGH.
github
If you are a git user, then simply cloning the repository will get you the latest code.
git clone git://github.com/seandavi/ngCGH.git
Alternatively, click the Download button and get the tarball or zip file.
In either case, change into the resulting directory and:
cd ngCGH python setup.py install
From PyPi
If you have easy_install in place, this should suffice for installation:
easy_install ngCGH
Usage
Usage is very simple:
usage: ngCGH [-h] [-w WINDOWSIZE] [-o OUTFILE] [-l LOGLEVEL] [-r REGIONS] [-t PROCESSES] normalbam tumorbam positional arguments: normalbam The name of the bamfile for the normal comparison tumorbam The name of the tumor sample bamfile optional arguments: -h, --help show this help message and exit -w WINDOWSIZE, --windowsize WINDOWSIZE The number of reads captured from the normal sample for calculation of copy number (default: 1000) -o OUTFILE, --outfile OUTFILE Output filename, default <stdout> (default: None) -l LOGLEVEL, --loglevel LOGLEVEL Logging Level, 1-30 with 1 being maximal logging and 30 being errors only [20] (default: 20) -r REGIONS, --regions REGIONS regions to which analysis should be restricted, either a bed file name or a single region in format chrN:XXX- YYY (default: None) -t PROCESSES, --threads PROCESSES parallelize over regions (or chromosomes) (default: 1)
Output
The output format is also very simple:
chr1 4851 52735 1000 854 -0.025120 chr1 52736 59251 1000 812 -0.097876 chr1 59251 119119 1000 876 0.011575 chr1 119120 707038 1000 1087 0.322924 chr1 707040 711128 1000 1016 0.225472 chr1 711128 711375 1000 1059 0.285275 chr1 711375 735366 1000 919 0.080709 chr1 735368 798455 1000 972 0.161600
Columns 1-3 describe the chromosome, start, and end for each pseudo-probe. The fourth column is the number of reads in the normal sample in the window while the fifth column represents the reads in the same genomic window from the tumor. The last column contains the median-centered log2 ratio between tumor and normal.
Convert from ngCGH to BioDiscovery Nexus
Included in the release is a script, convert2nexus, that takes as input the filename of a file created by ngCGH and converts it into a file that the Nexus CGH software from BioDiscovery can load for further analysis. The format looks like this:
Name Chromosome Start End PALZGU.cgh chr1_10004 chr1 10004 15735 -2.087921 chr1_15736 chr1 15736 69385 -2.670936 chr1_69386 chr1 69386 521687 -0.428244 chr1_523537 chr1 523537 726959 0.080269 chr1_726959 chr1 726959 808542 0.223047 chr1_808546 chr1 808546 809138 -1.186761
I presented a webinar on using ngCGH with BioDiscovery Nexus that you can view here.
Convert from Complete Genomics to BioDiscovery Nexus
There is now plenty of Complete Genomics data floating around. We are often interested in visualizing the somatic CNV data in Biodiscovery nexus. There is a script, cgi2nexus that takes a file typically named as “SomaticCnvDetailsDiploidBeta*” and converts to the file format noted above. Bzip2 (typical from CGI) are uncompressed on-the-fly.
Segmenting output
The cgh2seg script uses some sane defaults (at least for exomes) to the Circular Binary Segmentation algorithm as implemented in the DNAcopy Bioconductor package. The segmented results are centered around the mode of the density of the segmented values on a per-probe basis. The script will write the “Centrality parameter” to stderr when it completes.
The file format is:
ID chrom loc.start loc.end num.mark seg.mean 09 chr1 367695 82438842 2279 0.546541374526925 09 chr1 82778033 93082545 206 0.077841374526925 09 chr1 93205647 103965955 188 -0.913458625473075 09 chr1 104000621 104166584 4 -0.216558625473075 09 chr1 104342470 110014374 109 -0.948958625473075 09 chr1 110024223 110058480 4 -1.38295862547308
Methods
The pseudo-cgh algorithm employed by ngCGH takes as input two appropriately matched BAM files, typically from a tumor and a matched normal. Genomic windows are defined by reading blocks of a fixed number of reads (default 1000 reads) in the normal sample. Within each defined genomic window, the number of reads in the tumor is quantified. For each genomic window, a ratio is made between the number of reads in the tumor and the number of reads in the normal. Finally, a log2 transformation is applied to each ratio and the entire vector of the results is then centered by subtracting the median.
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