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AddReferenceGenome.md

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Configuring reference genome

For GC correction and 1000 genome strict mask filtering CNVpytor uses information related to the reference genome. With installation two reference genomes are available: hg19 (GRCh37) and hg38 (GRCh38).

If we want to use CNVpytor for other species or different reference genome for human first we have to create GC and mask file (optional).

In this example we will configure mouse reference genome MGSCv37.

To create GC file we need sequence of the reference genome in fasta.gz file:

> cnvpytor -root MGSCv37_gc_file.pytor -gc ~/hg19/mouse.fasta.gz -make_gc_file

This command will produce MGSCv37_gc_file.pytor file that contains information about GC content in 100-base-pair bins.

For reference genomes where we have strict mask in the same format as 1000 Genomes Project strict mask, we can create mask file using command:

> cnvpytor -root MGSCv37_mask_file.pytor -mask ~/hg19/mouse.strict_mask.whole_genome.fasta.gz -make_mask_file

If we do not have mask file, we can skip this step. Mask file contains information about regions of the genome that are more accessible to next generation sequencing methods using short reads. CNVpytor uses P marked positions to filter SNP-s and read depth signal. If reference genome configuration does not contain mask file, CNVpytor will still be fully functional, apart from the filtering step. You may also generate your own mask file by creating fasta file that contains character "P" if corresponding base pair passes the filter and any character different than "P" if not.

Now, we will create example_ref_genome_conf.py file containing list of chromosomes and chromosome lengths:

import_reference_genomes = {
    "mm9": {
        "name": "MGSCv37",
        "species": "Mus musculus",
        "chromosomes": OrderedDict(
            [("chr1", (197195432, "A")), ("chr2", (181748087, "A")), ("chr3", (159599783, "A")),
            ("chr4", (155630120, "A")), ("chr5", (152537259, "A")), ("chr6", (149517037, "A")),
            ("chr7", (152524553, "A")), ("chr8", (131738871, "A")), ("chr9", (124076172, "A")),
            ("chr10", (129993255, "A")), ("chr11", (121843856, "A")), ("chr12", (121257530, "A")),
            ("chr13", (120284312, "A")), ("chr14", (125194864, "A")), ("chr15", (103494974, "A")),
            ("chr16", (98319150, "A")), ("chr17", (95272651, "A")), ("chr18", (90772031, "A")),
            ("chr19", (61342430, "A")), ("chrX", (166650296, "S")), ("chrY", (15902555, "S")),
            ("chrM", (16299, "M"))]),
        "gc_file":"/..PATH../MGSCv37_gc_file.pytor",
        "mask_file": "/..PATH../MGSCv37_mask_file.pytor"
    }
}

Last line can be skipped, if there is no mask file. Letter next to chromosome length denote type of a chromosome: A - autosome, S - sex chromosome, M - mitochondria.

To use CNVpytor with new reference genome us -conf option in each cnvpytor command, e.g.

cnvpytor -conf REL_PATH/example_ref_genome_conf.py -root file.pytor -rd file.bam

CNVpytor will use chromosome lengths from alignment file to detect reference genome. However, if you configured reference genome after you had already run -rd step you could assign reference genome using -rg:

cnvpytor -conf REL_PATH/example_ref_genome_conf.py -root file.pytor -rg mm9

To avoid typing -conf REL_PATH/example_ref_genome_conf.py" each time you run cnvpytor, you can create an bash alias or make configuration permanent by copying example_ref_genome_conf.py to ~/.cnvpytor/reference_genomes_conf.py.

We would like to encourage you to send us configuration, gc and mask file and we would be glad to include it into the CNVpytor code. Or, even better, fork the repository on GitHub, add configuration in cnvpytor/genome.py, data files in cnvpytor/data and create a pull request.