Using DNA sequencing to assess dietary species richness

摘要：有效的饮食控制措施对监测和改善人类健康至关重要。一个简单 衡量标准是饮食多样性（即，在一段时间内吃的不同食物的数量），这可以作为 营养不良的标志然而，对饮食多样性的调查受到当前评估的限制。 依赖于饮食自我报告的技术。调查饮食多样性的另一种方法是使用 生物标志物或饮食的生化指标。然而，一种饮食生物标志物， 饮食多样性有待发展。在这里，我们将开发和验证一种新技术，称为DNA 元条形码，用于枚举个体食用的膳食植物和动物物种的数量（膳食 物种丰富度）。我们的方法建立在生态学家研究复杂摄食的概念上 在野生动物种群中的实践，即饮食DNA在消化中存活，并且可以在粪便中检测到 使用高通量DNA测序。我们的初步研究支持这种方法的承诺：我们有 成功地扩增和测序了1000多个人类粪便中的100多个饮食物种 在多个国家收集的样本。我们的试点工作也表明， 通过DNA元条形码和基于调查的饮食多样性指数捕获的饮食物种数量， 质量.为了进一步建立DNA元条形码作为饮食多样性的可靠和有用的标记，我们的团队 粪便基因组学、营养流行病学和生物统计学方面的专家将追求两个特定目标。一是 优化用于评估膳食动物物种摄入量的DNA元条形码。这一目标将建立在我们现有的 膳食植物的元条形码分析的方案。我们将在良好的条件下进行实验- 使用未加工和加工食品的混合物控制实验室设置。然后我们将测试我们最有希望的 使用从接受控制喂养的人类队列收集的粪便样品的储存库的方案。 其次，我们将测试的有效性和实用性，测量饮食多样性使用DNA元条码。这一目标将 将该技术应用于：1）来自低收入家庭的主要是非洲裔美国人/西班牙裔青年的队列 参加了一项肥胖治疗研究; 2）来自五个国家的1,000名非洲裔人组成的队列 不同的饮食习惯和心脏代谢疾病的风险。我们将利用这些研究来验证， metabarcoding物种丰富度反映了现有的饮食多样性的措施， 饮食摄入量调查。这些真实世界的队列将进一步使我们能够将元条形码数据整合到 代谢疾病风险模型，检查元条形码结果的时间趋势，并确定潜在的 食物物种丰富度的地理和社会经济决定因素。

ABSTRACT: Valid measures of diet are essential for monitoring and improving human health. One simple measure is dietary diversity (i.e., the number of different foods eaten over a period of time), which can serve as a marker of nutritional inadequacies. Surveys of dietary diversity, however, are limited by current assessment techniques that rely on dietary self-report. Another approach to surveying dietary diversity would be to use biomarkers or biochemical indicators of diet. However, a dietary biomarker that specifically captures overall dietary diversity has yet to be developed. Here, we will develop and validate a new technique, known as DNA metabarcoding, for enumerating the number of dietary plant and animal species individuals consume (dietary species richness). Our approach builds on a conceptual insight made by ecologists studying complex feeding practices in wild animal populations, which is that dietary DNA survives digestion and can be detected in stool using high-throughput DNA sequencing. Our preliminary studies support the promise of this approach: we have successfully amplified and sequenced more than a hundred dietary species from over a thousand human stool samples collected across multiple countries. Our pilot work has also shown significant correlations between the number of dietary species captured by DNA metabarcoding and survey-based indices of dietary diversity and quality. To further establish DNA metabarcoding as a reliable and useful marker of dietary diversity, our team of experts in fecal genomics, nutritional epidemiology, and biostatistics will pursue two Specific Aims. First, we will optimize DNA metabarcoding for assessing intake of dietary animal species. This Aim will build on our existing protocols for metabarcoding analysis of dietary plants. We will perform bench-top experiments under well- controlled lab settings using mixtures of intact and processed foods. We will then test our most promising protocols using a repository of stool samples collected from human cohorts undergoing controlled feeding. Second, we will test the validity and utility of measuring dietary diversity using DNA metabarcoding. This Aim will apply the technique to: 1) a cohort of primarily African-American/Hispanic youth from low-income families enrolled in a study of obesity treatment; and, 2) a cohort of 1,000 individuals of African descent from five countries with varying dietary habits and cardiometabolic disease risk. We will use these studies to validate that metabarcoding species richness reflects existing measures of dietary diversity measured by recall-based surveys of dietary intake. These real-world cohorts will further allow us to integrate metabarcoding data into models of metabolic disease risk, examine temporal trends in metabarcoding results, and identify potential geographic and socioeconomic determinants of dietary species richness.