Amir Amnon, McDonald Daniel, Navas-Molina Jose A, Debelius Justine, Morton James T, Hyde Embriette, Robbins-Pianka Adam, Knight Rob
Department of Pediatrics, University of California San Diego, La Jolla, California, USA.
Department of Computer Science, University of Colorado Boulder, Boulder, Colorado, USA.
mSystems. 2017 Mar 7;2(2). doi: 10.1128/mSystems.00199-16. eCollection 2017 Mar-Apr.
The use of sterile swabs is a convenient and common way to collect microbiome samples, and many studies have shown that the effects of room-temperature storage are smaller than physiologically relevant differences between subjects. However, several bacterial taxa, notably members of the class , grow at room temperature, sometimes confusing microbiome results, particularly when stability is assumed. Although comparative benchmarking has shown that several preservation methods, including the use of 95% ethanol, fecal occult blood test (FOBT) and FTA cards, and Omnigene-GUT kits, reduce changes in taxon abundance during room-temperature storage, these techniques all have drawbacks and cannot be applied retrospectively to samples that have already been collected. Here we performed a meta-analysis using several different microbiome sample storage condition studies, showing consistent trends in which specific bacteria grew (i.e., "bloomed") at room temperature, and introduce a procedure for removing the sequences that most distort analyses. In contrast to similarity-based clustering using operational taxonomic units (OTUs), we use a new technique called "Deblur" to identify the exact sequences corresponding to blooming taxa, greatly reducing false positives and also dramatically decreasing runtime. We show that applying this technique to samples collected for the American Gut Project (AGP), for which participants simply mail samples back without the use of ice packs or other preservatives, yields results consistent with published microbiome studies performed with frozen or otherwise preserved samples. In many microbiome studies, the necessity to store samples at room temperature (i.e., remote fieldwork) and the ability to ship samples without hazardous materials that require special handling training, such as ethanol (i.e., citizen science efforts), is paramount. However, although room-temperature storage for a few days has been shown not to obscure physiologically relevant microbiome differences between comparison groups, there are still changes in specific bacterial taxa, notably, in members of the class , that can make microbiome profiles difficult to interpret. Here we identify the most problematic taxa and show that removing sequences from just a few fast-growing taxa is sufficient to correct microbiome profiles.
使用无菌拭子是采集微生物组样本的一种方便且常见的方法，许多研究表明，室温保存的影响小于个体间生理上的相关差异。然而，有几种细菌类群，特别是 纲的成员，会在室温下生长，有时会混淆微生物组的结果，尤其是在假定稳定性的情况下。尽管比较基准测试表明，包括使用 95%乙醇、粪便潜血试验（FOBT）和 FTA 卡以及 Omnigene - GUT 试剂盒在内的几种保存方法，可减少室温保存期间分类群丰度的变化，但这些技术都有缺点，且不能追溯应用于已经采集的样本。在这里，我们使用几个不同的微生物组样本存储条件研究进行了一项荟萃分析，显示出特定细菌在室温下生长（即“大量繁殖”）的一致趋势，并引入了一种去除最扭曲分析的序列的程序。与使用操作分类单元（OTU）进行基于相似性的聚类不同，我们使用一种名为“Deblur”的新技术来识别与大量繁殖的分类群相对应的精确序列，大大减少了假阳性，同时也显著缩短了运行时间。我们表明，将这项技术应用于为美国肠道项目（AGP）采集的样本，该项目的参与者只需将样本邮寄回去，无需使用冰袋或其他防腐剂，其结果与对冷冻或其他保存样本进行的已发表微生物组研究一致。在许多微生物组研究中，在室温下保存样本的必要性（即野外实地工作）以及在不使用需要特殊处理培训的危险材料（如乙醇）的情况下运送样本的能力（即公民科学活动）至关重要。然而，尽管已表明室温保存几天不会掩盖比较组之间生理上相关的微生物组差异，但特定细菌类群仍有变化，特别是 纲的成员，这可能使微生物组图谱难以解读。在这里，我们确定了最成问题的分类群，并表明仅从少数快速生长的分类群中去除序列就足以校正微生物组图谱。
