Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing.
Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing.
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DOI：
10.3390/genes12010106
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发表时间：
2021-01-16
期刊：
影响因子：
3.5
通讯作者：
Castro-Wallace SL
中科院分区：
文献类型：
--
作者：
Stahl-Rommel S;Jain M;Nguyen HN;Arnold RR;Aunon-Chancellor SM;Sharp GM;Castro CL;John KK;Juul S;Turner DJ;Stoddart D;Paten B;Akeson M;Burton AS;Castro-Wallace SL
在过去的二十年里，国际空间站 (ISS) 的微生物监测一直依赖于依赖培养的方法，这些方法需要返回地球进行分析。这有许多限制，其中最重要的是对可培养生物体检测的偏见以及样本收集和地面分析之间固有的延迟。近年来，便携式且易于使用的分子工具，例如 Oxford Nanopore Technologies 的 MinION™ 测序仪和 miniPCR bio 的 miniPCR™ 热循环仪，已在国际空间站上得到验证。在这里，我们报告了拭子到测序仪方法的开发、验证和实施，该方法为国际空间站上的实时微生物分析提供了独立于培养的解决方案。方法开发的重点是对在低生物量环境中收集的拭子进行分析，该环境中设施资源有限，并且对允许的流程和试剂进行严格控制。 ISS 优化程序包括从拭子尖端酶促 DNA 提取、基于珠子的纯化、改变缓冲液以及使用 miniPCR 和 MinION。验证是通过广泛的地面评估进行的，比较当前标准的依赖于培养的方法和新开发的不依赖于培养的方法。两种方法之间观察到相似的微生物分布；然而，正如预期的那样，独立于培养物的数据揭示了具有更大多样性的微生物特征。协议优化和验证分别是在 NASA 极端环境任务操作 (NEEMO) 模拟任务 21 和 22 期间建立的。从模拟测试中获得的独特微生物特征在极端环境下验证了拭子到测序仪的方法。最后，两名机组人员在国际空间站上进行了四次独立的拭子到测序仪实验。从 ISS 拭子中鉴定出的微生物与历史培养数据一致，主要由常见的人类相关微生物组成。这种简化的方法经过简化，非常易于使用，可供未经训练的机组人员在极端环境中完成，从而在未来的任务带我们超越近地轨道时实现环境和人类健康的实时诊断。
For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies’ MinION™ sequencer and miniPCR bio’s miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.
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影响因子：
4.2
作者：
Brooks JP;Edwards DJ;Harwich MD Jr;Rivera MC;Fettweis JM;Serrano MG;Reris RA;Sheth NU;Huang B;Girerd P;Vaginal Microbiome Consortium;Strauss JF 3rd;Jefferson KK;Buck GA
通讯作者：
Buck GA
影响因子：
4.6
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Castro-Wallace SL;Chiu CY;John KK;Stahl SE;Rubins KH;McIntyre ABR;Dworkin JP;Lupisella ML;Smith DJ;Botkin DJ;Stephenson TA;Juul S;Turner DJ;Izquierdo F;Federman S;Stryke D;Somasekar S;Alexander N;Yu G;Mason CE;Burton AS
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Burton AS
影响因子：
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作者：
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通讯作者：
ROBERTS, RB
影响因子：
3.6
作者：
Castro, VA;Thrasher, AN;Pierson, DL
通讯作者：
Pierson, DL
影响因子：
4.7
作者：
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