VCF

The main output of Rastair is a VCF file. It contains all metrics that Rastair calculates, either for all variants, or only for CpG sites.

BCF output, compression

VCF files are text-based and can be quite large, especially for whole-genome sequencing data. Rastair can also output BCF files (binary VCF format) which are more compact and faster to read. Alternatively, it can compress the VCF file transparently using bgzip. All formats can be read by bcftools, just like regular VCF files.

By specifying the file extension (.vcf, .bcf, or .vcf.gz) Rastair will automatically detect which format to write.

Fields

By default, only a few fields are include. You can enable more using the --vcf-info-fields and --vcf-info-fields flags with comma-separated field names. See VCF Fields for a detailed description of the fields in the VCF output.

Methylation information

The estimated $\beta$ value is encoded in the VCF file through the M5mC FORMAT field which is provided at all positions where CPG or CPGnovo are set. We include all CpG positions in the reference, whether they overlap a variant or not. Where they do not overlap a variant, the ALT is set to ., aka the missing value.

While rare, it is possible to have more than one methylation call at one position. This can occur in CCG positions where the middle C is also a C>G SNP. This leads to two alleles, one where the middle position is a G and the other where it is a C. Each could be methylated at different levels. In these cases, the M5mC value will be a comma-separated list.

Quality scores

Quality scores in the vcf output reflect the ML-derived probability that a variant is "true". When we train the ML model, we calibrate it against a validation dataset and estimate the fraction of true SNPs called at every score cutoff. Based on this, we can fit a regression ("Platt scaling") that converts the score into a probability $p$ that a SNP call is true. For the quality score, we encode the inverse as Phred: $-10\ast lo{g}_{10}(1-p)$. For methylated positions where no variants were observed, we record the maximum Phred, ie. 99.