Sequence data obtained from each sample was subjected to standard Illumina QC procedures and 20 million reads per sample was subjected to detailed analysis for enrichment, quality, content, and coverage. Each dataset was analysed independently by mapping sequence reads to the 3D7 reference genome using BWA [13 (link)]. SAMtools [14 (link)] was used to generate coverage statistics from the BWA mapping output. For enrichment analysis, the number of reads mapping to either host, or P. falciparum reference sequences was counted. For genotype and concordance analysis, variant calls were generated using SAMtools mpileup (V0.1.1.19; with the following parameters: -DSV -C50 -m2 -F0.0005 -d 10,000 -gu) and bcftools (V0.1.17; with the following parameters: -p 0.99 -vcgN). A list of 1,241,840 (1.2 million) high-quality single-nucleotide polymorphism (SNP) positions, which were not filtered by gene class or region, but on individual properties of SNPs (such as uniqueness of the surrounding region and within an exon) [15 (link), 16 ] was used. In silico genotyping of both the DBS (sWGA) and VB (leucodepleted and unamplified) samples was performed using mpileup to count alleles present in at least five reads (alleles with less than five reads were discarded). Although P. falciparum is haploid, it is common to find heterozygous calls due to the presence of multiple clonal infections in the same host. In order to genotype heterozygous sites, the 5/2 rule was applied, which requires at least two reads in both reference and alternative alleles, and the sum of both has to be higher than five reads [15 (link)]. SNP call concordance analysis between matching DBS and VB samples was performed on sequenced data targeting SNPS present in the core genome as well as key malaria drug resistance genes, such as crt (K76T involved in chloroquine resistance) [17 (link)], dhfr (N51I, involved in pyrimethamine resistance) [18 (link)], dhps (A581G, involved in sulfadoxine resistance) [19 (link)], mdr1 (N86Y, involved in multiple drugs including mefloquine) [20 (link)], and kelch13 (C580Y, involved in artemisinin resistance) [1 (link)].
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