(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 <em>P. falciparum</em> diversity throughout treatment in a clinical drug trial <strong>Table S1. List of PCR and deep sequencing primers.</strong> 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. <strong>Table S2</strong><strong>. </strong><strong>The relative frequencies of each </strong><strong>ama1</strong><strong> variant and the number of samples with each variant<em>. </em></strong>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). <strong>Figure S1</strong><strong>. </strong><strong>Performance of TADS on the sequencing controls made up of a mixture of laboratory isolates.</strong> 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 <em>ama1</em>, sequencing failed for replicate 6 of control set 1, while for <em>mdr1</em>, 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%. <strong>Figure S2. Heatmaps of the successfully PCR amplified and sequenced samples for </strong><strong><em>ama1</em> (A) and </strong><strong><em>mdr1</em> (B).</strong> 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 (&gt; 5,000), moderate (100-5,000) and low (&lt; 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. <strong>Figure S3</strong><strong>. The mean complexity of infection (COI) by AMA1 throughout treatment.</strong> 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 (&lt;5) in the post-treatment samples. The black dots represent the COI per sample.本补充数据集配套如下研究手稿：《临床药物试验中采用ama1与mdr1靶向扩增子深度测序追踪治疗过程中宿主体内恶性疟原虫（*Plasmodium falciparum*）多样性》 **表S1. PCR与深度测序引物列表。**本表列出了用于深度测序的正向与反向引物，其中粗体字体标注的为MID标签，常规字体标注的为正向引物。 **表S2. 各ama1变异体的相对频率及携带各变异体的样本数。**本文展示了33种AMA1变异体在治疗前与治疗后样本（n=330）中的相对频率（%），以33个氨基酸序列形式呈现。频率计算公式为：单倍型读段数除以总获取读段数（116,187,131）。 **图S1. 实验室分离株混合构建的测序对照中TADS的检测性能。**每组对照设置6次重复，以应对潜在的扩增或测序失败风险，确保获得充足的对照数据。实验室对照的中位读段深度分别为5658（范围：4310~12603）与658（范围：-1676）。横轴为5组混合样本的重复编号，取值范围为1至6；纵轴为各变异体在所有重复样本中的占比。针对ama1（子图A），共检测到2种变异体（3D7与Dd2）；针对MDR1（子图B），分别扩增Dd2拷贝I、Dd2拷贝II与3D7后，共检出YY、FY与NY三种变异体。针对ama1，对照1组的重复6出现测序失败；针对mdr1，对照3组的重复2与6、对照4组的重复1与6，以及对照5组的重复1与5均发生测序失败。在对照4组的MDR1检测中，Dd2拷贝II（86F, 184Y）未被检出，推测原因是其浓度过低，未被本次重复实验捕获。基于本对照混合样本，本研究可检出的最低变异体频率为0.5%。 **图S2. 成功经PCR扩增并测序的ama1（A）与mdr1（B）样本热图。**行代表研究受试者，列代表采样时间（小时）。成功测序的样本以绿色标注，PCR扩增失败的样本以红色标注，测序失败的样本以黄色标注。时间点Rec代表非计划随访，此时采集了复发样本。带有“-”的无阴影区域代表未采集样本的时间点。每个时间点的成功测序样本数n标注于各子图的最后一行。子图C中的表格展示了按原虫血症水平分组的样本：高（>5000个寄生虫/微升）、中（100~5000个寄生虫/微升）及低（<100个寄生虫/微升）。0~12小时采集的多数样本原虫血症水平较高，18~30小时采集的样本原虫血症水平中等，而30小时后采集的样本原虫血症水平以低水平为主。 **图S3. 治疗全程基于AMA1的平均感染复杂度（Complexity of Infection, COI）。**平均感染复杂度（红色菱形）在基线（0小时）至72小时期间保持稳定（介于1.5~2之间），后续因治疗后样本量较小（n<5）而出现波动。黑色圆点代表单份样本的感染复杂度。