Sequencing human genetic variants associated with malarial disease in Northeast Tanzania. Human amplicon sequencing in Northeast Tanzania

Malaria has exhibited the strongest known selective pressure on the human genome in recent history and is the evolutionary driving force behind genetic conditions, such as sickle-cell disease, glucose-6-phosphatase deficiency, and countless other erythrocyte defects. Genomic studies (e.g., The 1000 Genomes project) have provided an invaluable baseline for human genetics, but, with an estimated two thousand ethno-linguistic groups thought to exist across the African continent, our understanding of the genetic differences between indigenous populations and their implications on disease is still limited. Low-cost sequencing-based approaches have made it possible to target molecular markers and genes of interest. Here we demonstrate the versatility of custom dual-indexing technology and Illumina next generation sequencing to generate a genetic profile of human polymorphisms associated with malaria pathology. For 100 individuals diagnosed with severe malaria in Northeast Tanzania, variants were successfully characterised on the haemoglobin subunit beta (HBB), glucose-6-phosphate dehydrogenase (G6PD), atypical chemokine receptor 1 (ACKR1) genes, and the intergenic Dantu genetic blood variant, then validated using pre-existing genotyping data. High sequencing coverage was observed across all amplicon targets and variants across HBB, G6PD, ACKR1, and the Dantu blood variant were identified at frequencies previously observed within this region of Tanzania. Sequencing data exhibited high concordance rates to pre-existing genotyping data, resulting in a minimum calling accuracy of 97%.

疟疾是近代以来已知对人类基因组施加最强选择压力的病原体，亦是镰状细胞病、葡萄糖-6-磷酸酶缺乏症（glucose-6-phosphatase deficiency）等诸多红细胞相关遗传缺陷的进化驱动力。基因组学研究（例如千人基因组计划（1000 Genomes Project））为人类遗传学研究提供了宝贵的基准数据集，但非洲大陆据估计存在两千余个民族语言群体，当前我们对本土人群间的遗传差异及其对疾病的影响仍存在认知局限。基于测序的低成本技术方案，使得靶向目标分子标记与功能基因成为可能。本研究展示了定制双索引技术与Illumina下一代测序（Illumina next generation sequencing）的多功能性，可用于构建与疟疾病理相关的人类多态性遗传图谱。针对坦桑尼亚东北部100名确诊重症疟疾的受试者，本研究成功对血红蛋白亚基β（haemoglobin subunit beta, HBB）、葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase, G6PD）、非典型趋化因子受体1（atypical chemokine receptor 1, ACKR1）基因以及基因间区Dantu血型遗传变异完成了变异分型，并通过已有的基因分型数据完成了验证。所有扩增子靶点均实现了较高的测序覆盖度，且在HBB、G6PD、ACKR1基因及Dantu血型变异中检出的变异频率，与此前坦桑尼亚该地区的报道一致。测序数据与已有的基因分型数据具有极高的一致性，变异检出准确率最低可达97%。