Doctoral Dissertation Research: Impact of Various Storage Temperatures andTtime on Microbiome of Forensically Relevant Biological Samples

法医相关体液的确认鉴定是法医调查的基本组成部分，因为这些信息可以帮助调查人员重建犯罪现场，并解决诸如性侵犯案件中经常遇到的混合情况。目前大多数体液鉴定方法都是推测性的，而且样本消耗量对于法医案件工作来说并不理想。为了使新方法被社会所接受，方法应该是健壮的、可靠的、敏感的和对每种体液的确认性的，同时随着时间的推移产生稳定的结果。本研究旨在确定人体生物样品的短期和长期储存条件如何影响与这些样品相关的微生物群落结构，并确定这些条件是否影响新开发的基于微生物特征的体液鉴定方法的准确性。生物样品的储存规则是在没有考虑微生物证据的情况下制定的，因此确定这些样品在各种储存时间和温度下是否稳定非常重要。该项目将1)观察每种体液的微生物特征，以确定在不同温度下长时间储存后其成分是否存在显著差异；2)确定这些储存条件是否会影响先前开发的体液鉴定方法的准确性。10个个体的样本将在4种不同温度（-20°C、4°C、25°C、37°C）下保存3个储存期（30天、60天、90天）。为了评估这些变化，16S rDNA双索引高通量测序将使用MiSeq FGx测序平台进行。数据分析将按照MiSeq母亲SOP进行。将评估所有温度/时间组之间细菌特征的变化。先前开发的集合子空间分类和l1范数正则化分类方法将使用获得的细菌结构进行测试，以确定存储条件如何影响分类准确性。这项研究的结果将有助于简化以前开发的体液鉴定方法，并将为标准操作程序提供额外的建议。这不仅对法医科学界有用，而且可以促进整个微生物研究的稳定性、标准化和流动性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The confirmatory identification of forensically relevant body fluids is a fundamental part to forensic investigations as this information can aid investigators in crime scene reconstruction and resolving mixtures such as those often encountered in sexual assault cases. The majority of current body fluid identification methods are presumptive, and the sample consumption is not ideal for forensic casework. For new methods to be accepted into the community, methods should be robust, reliable, sensitive, and confirmatory to each body fluid while producing stable results over time. This study aims to determine how short- and long-term storage conditions of human biological samples impact the microbial community structures associated with these samples, and to determine whether these conditions impact the accuracy of a newly developed microbial signature-based method for the body fluid identification. Biological sample storage regulations were created without microbial evidence in mind, and it is therefore important to determine whether these samples are stable under various storage times and temperatures. This project will 1) look at the microbial signatures of each body fluid to determine whether there are significant differences in their compositions once stored at various temperatures at extended periods of time and 2) determine whether these storage conditions impact the accuracy of the previously developed body fluid identification method. Samples from ten individuals will be stored at four different temperatures (-20°C, 4°C, 25°C, 37°C) for 3 storage periods (30 days, 60 days, 90 days). To evaluate these changes, 16S rDNA dual-index high throughput sequencing will be performed using the MiSeq FGx sequencing platform. Data analysis will follow the mothur MiSeq SOP. The variation in bacterial signatures will be assessed between all temperature/time groups. Ensemble subspace classification and L1-norm regularized classification methods previously developed will be tested using the bacterial structures obtained to determine how storage conditions may impact the classification accuracy. The findings from this study will be useful in streamlining the previously developed body fluid identification method and will provide additional suggestions for standard operating procedures. This is not only useful for the forensic science community but can contribute to stability, standardization, and mobility of microbial research as a whole.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.