(Extended Data) Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

These extended data accompany the manuscript: Targeted Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Table S1. List of PCR and deep sequencing primers. This table shows the list of forward and reverse primers used for deep sequencing. In boldface are the MID tags while in the regular face are the forward primers.

Table S2. The relative frequencies of each ama1 variant and the number of samples with each variant. The relative frequencies (%) of the 33 AMA1 variants in pre-and post-treatment samples (n = 330) are shown as a 33 amino acid sequence. The frequencies were calculated by dividing the number of reads of each haplotype by the total number of reads obtained (116,187,131).

Figure S1. Performance of TADS on the sequencing controls made up of a mixture of laboratory isolates. Each control set was done in 6 replicates to ensure sufficient control data in the event of potential amplicon or sequencing failure. The median read depth in the lab controls was 5658 (range 4,310 – 12,603) and 658 (- 1,676). The x-axis represents the replicate identifier across the five mixtures, starting from 1 to 6 while the y-axis represents the proportions of each variant across all replicates. For ama1 (A), 2 variants (3D7 and Dd2) were detected whereas in MDR1 (B), 3 variants were detected YY, FY and NY following amplification of Dd2 Copy I, Dd2 Copy II and 3D7, respectively. For ama1, sequencing failed for replicate 6 of control set 1, while for mdr1, sequencing failed for replicate 2 and 6 of control set 3, replicates 1 and 6 of control set 4 and replicates 1 and 5 of control set 5. Under the MDR1 control set 4, the Dd2 copy II (86F, 184Y) was not identified possibly due to having very low concentrations that were not picked up in this replicate. Based on our control mixtures, the minimum variant frequency we were able to detect was 0.5%.

Figure S2. Heatmaps of the successfully PCR amplified and sequenced samples for ama1 (A) and mdr1 (B). The rows represent the study participants while the columns represent time in hours. Successfully sequenced samples are shown in green, those that failed PCR in red and those that failed sequencing are in yellow. The timepoint Rec, represent unscheduled visits where a recurrent sample was collected. The unshaded areas with ‘-‘ are timepoints where samples were not collected. For each time-point, the number of samples successfully sequenced, n, is indicated in the last row of each panel. The table in panel C shows the groupings of samples based on parasitemia, high (> 5,000), moderate (100-5,000) and low (< 100 parasites per microlitre). Many samples collected between 0h-12h had high parasitemia, samples collected between 18h-30h had moderate parasitemia while samples collected after 30h were primarily of low parasitemia.

Figure S3. The mean complexity of infection (COI) by AMA1 throughout treatment. The mean COI (red diamonds) appeared to be stable (between 1.5 - 2) from baseline (0h) up to 72h and thereafter fluctuates due to the small sample sizes (<5) in the post-treatment samples. The black dots represent the COI per sample.