Table 3.

Definitions of heterozygous base calls used to classify mixed infection

Journal article	Mixed infections or microevolution	Definition of heterozygous base call
Bryant et al. [30]	Mixed infection	Mixed base positions were identified at sites where more than one base had been identified in a single sample, where each allele was supported by at least 5 % of reads (minimum read depth of four). Included only positions without strand bias (p >0.05), had coverage within the normal range, mapping quality score greater than 50 and base quality scores greater than 30. Sites within 200 base pairs of other heterozygous sites were discounted because of the possibility that they might have been caused by a mapping error. More than 80 heterozygous base calls defined a mixed infection
Guerra-Assunção et al. [18]	Mixed infection	Sample genotypes were called using the majority allele (minimum frequency 75 %) in positions supported by at least 20-fold coverage; otherwise they were classified as missing (thus ignoring heterozygous calls). We excluded samples with >15 % missing genotype calls, to remove possible contaminated or mixed samples or technical errors
Guerra-Assunção et al. [29]	Mixed infection	A position was classified as heterozygous if >1 allele accounted for ≥30 % of the reads (and there were >30 reads). More than 140 heterozygous positions in one sample classified as mixed infection
Kato-Maeda et al. [26]	Mixed infection	Mixed infection was identified when there was a heterozygous base call: 38 % of reads supported the variant; the rest supported reference
Luo et al. [16]	Microevolution	Kept only the calls in which the coverage was ten and the less frequent allele was supported by at least five high-quality reads, as reliable calls. Presence of mixed base calls could indicate microevolution in that patient
Pérez-Lago et al. [31]	Mixed infection	Less frequent nucleotide was supported by five reads
Walker et al. [21]	Microevolution	Suggestive of ‘sub-populations’; i.e. microevolution