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Allele calling was achieved by comparison with the allelic ladder provided in the kit, helped by GeneMapper software (version 3.2).
Subsequent gapped read alignment and variant calling was achieved using Bioscope 1.3.1 (Life Technologies).
Variant calling was achieved using FreeBayes (http://arxiv.org/abs/1207.3907) to produce VCF files, one for each of the sequenced accessions.
The power of covariance was also harnessed in a recent study of HCV early evolution, where SNV calling was achieved by parsing haplotypes reconstructed using probabilistic clustering performed by the program ShoRAH [ 6].
Automatic allele calling was achieved using the Illumina Genome Studio software v2011.1 with a GeneCall threshold of 0.20 and checking the output visually as well for the confirmation of cluster specificity.
In the first (CcRAD1), a comprehensive list of putative SNPs and 1-2 bp indels was populated with a minimum coverage of 6x as threshold prior to uploading to a Microsoft Access relational database; and for the second (CcRAD2), "fully informative" SNPs were defined when a minimum of 1-read allele calling was achieved for each of the three samples.
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However, our results show that more accurate SNV calling is achieved if the expected number of forward reads is modelled using a beta-binomial read distribution, compared to either a binomial distribution or applying Fisher's exact test.
The results showed that more than a 90% reduction in high-fidelity function calls was achieved when compared to direct high-fidelity model optimization using a pattern-search algorithm.
More than a 90% reduction in high-fidelity function calls is achieved when compared to direct highfidelity model optimization.
At the same time, the values of excess attenuation were lower for soft calls than for broadcast calls at most distances, which suggests that the short transmission of soft calls is achieved mostly due to their low and narrow frequency ranges, promoting their masking by ambient noise.
This increased the overlap of genotype calls to dbSNP v137, suggesting that lower false-positive SNP calls were achieved (Supplementary Table S4).
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