The microbiome-gut-brain-axis field is multidisciplinary, benefiting from the expertise of microbiology, ecology, psychiatry, computational biology, and epidemiology amongst other disciplines. As the field matures and moves beyond a basic demonstration of its relevance, it is critical that study design and analysis are robust and foster reproducibility. In this companion piece to Bugs as Features (Part 1), we present techniques from adjacent and disparate fields to enrich and inform the analysis of microbiome-gut-brain-axis data. Emerging techniques built specifically for the microbiome-gut-brain axis are also demonstrated. All of these methods are contextualised to inform several common challenges: how do we establish causality? How can we integrate data from multiple 'omics techniques? How might we account for the dynamicism of host-microbiome interactions? This perspective is offered to experienced and emerging microbiome scientists alike, to assist with these questions and others, at the study conception, design, analysis and interpretation stages of research.

翻译：微生物组-肠-脑轴领域具有多学科交叉特性，受益于微生物学、生态学、精神病学、计算生物学和流行病学等学科的专长。随着该领域日趋成熟并超越基础相关性验证阶段，研究设计与分析的稳健性及促进可重复性变得至关重要。作为《微生物作为特征（第一部分）》的姊妹篇，我们展示了来自邻近与不同学科的技术，以丰富和指导微生物组-肠-脑轴数据分析。同时，还介绍了专门为微生物组-肠-脑轴构建的新兴技术。所有这些方法均被置于具体背景中，以应对若干常见挑战：如何建立因果关系？如何整合来自多种组学技术的数据？如何考虑宿主-微生物相互作用的动态性？本文旨在为经验丰富的微生物组科学家及该领域的新兴研究者提供视角，从研究构思、设计、分析到解释阶段，协助应对这些问题及其他挑战。