Illustration of the ROVER principles. The lower panel represents contiguous sections of a reference sequence (e.g. a specific human genome build). Numbered ‘F’ and ‘R’ pairs denote primer co-ordinates used in conjunction with mapped read starting co-ordinates to guide clipping (dotted boxes). Shaded boxes represent regions under analysis. Arrows represent reads of read-pairs. ‘X’s denote positions different from the reference sequence. Only when both reads of a read-pair concur can the read-pair contribute to genetic variant calling for a given position (circled). This contribution to a read-pair pile-up allows variant calling using thresholds for minimum number of read-pairs and the proportion of read-pairs containing the variant.