Precautions against numt in the other tiger subspecies had been previously noted [31]; among the outgroups, primers had been used for sequences conserved among the Felidae, while the conservation of open reading frames in the mtDNA genes was an indication that numt had been avoided [14], [16].
This selection enabled us to assess the generality of our findings and identify any impacts of gene length, type (RNA vs. protein), function (housekeeping vs. not), or level of conservation on read-tree construction.
Comparison of its sequence to the functional NANOG gene's reading frame suggests that this apparent pseudogene remained functional after duplication and, therefore, was subject to selection-driven conservation of its reading frame, and that it may retain some functionality or that its loss of function may be evolutionarily recent.
This indeed is often the case for deep relationships and it is well known that coding mtDNA such as cox1 displays some advantage due to the conservation of the reading frame which usually provides unambiguous guidance for a global alignment [85].
Taken in the aggregate, these observations strongly support the hypothesis that NANOGP1 remained functional after duplication and, therefore, was subject to selection-driven conservation of its reading frame.
Before insertion of NANOGP4, the region on chromosome 12 containing NANOG underwent a duplication producing NANOGP1, which remained functional and subject to selection-driven conservation of its reading frame.
The greatest attrition in candidate SNPs, however, was based on SNP conservation between reads of a single germplasm (selecting 100% consistency within genotypes).
The greatest attrition, accounting for 55% of all in silico SNPs, was caused by lack of SNP conservation between sequence reads of a single genotype.
The distributions of reads in these windows were then manually inspected in Integrative Genome Viewer (IGV) [ 49] and cross referenced with genes, conservation and repetitive elements.
