Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags.
Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags.
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DOI：
10.1186/1471-2180-10-206
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发表时间：
2010-07-30
期刊：
影响因子：
4.2
通讯作者：
Bushman FD
中科院分区：
文献类型：
--
作者：
Wu GD;Lewis JD;Hoffmann C;Chen YY;Knight R;Bittinger K;Hwang J;Chen J;Berkowsky R;Nessel L;Li H;Bushman FD
人们对人类肠道微生物组在健康和疾病中的作用非常感兴趣，但最佳分析方法仍在开发中。在这里，我们提出了一项研究的方法，调查人类粪便中的细菌群落使用454/罗氏焦磷酸测序的16 S rRNA基因标签。我们分析了来自10个个体的粪便样本，并比较了储存、DNA纯化和序列获取的方法。为了评估重现性，我们比较了每个个体的单个粪便标本上相隔1 cm的样本。为了分析储存方法，我们比较了1）在-80 ° C下立即冷冻，2）在冰上储存24或3）48小时。对于DNA纯化方法，我们测试了三种商业试剂盒和在热苯酚中的珠击。由于不同的方法的变化进行了比较，使用两种方法的个人之间的变化-一个基于存在-不存在的信息细菌类群（未加权UniFrac）和其他考虑到它们的相对丰度（加权UniFrac）。在未加权分析中，与不同分析方法相关的变异相对较小，个体之间的变异占主导地位。在加权分析中，相当大的变化与纯化方法有关。特别值得注意的是使用热酚法提高了厚壁菌门序列的回收率。我们还进行了调查的影响，不同的454测序方法（FLX与钛）和不同的16 S rRNA可变基因片段的扩增。根据我们的研究结果，我们提出了从粪便样本中收集、处理和测序细菌16 S rDNA的建议--一些要点是：1）如果可行，在热苯酚中进行珠粒打浆或使用PSP试剂盒可以提高回收率; 2）可以根据实验便利性调整储存方法; 3）未加权（存在-不存在）比较受裂解方法的影响较小。
Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80°C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.
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影响因子：
12.3
作者：
Huse SM;Huber JA;Morrison HG;Sogin ML;Welch DM
通讯作者：
Welch DM
影响因子：
14.9
作者：
Ludwig, W;Strunk, O;Schleifer, KH
通讯作者：
Schleifer, KH
影响因子：
3.7
作者：
Claesson MJ;O'Sullivan O;Wang Q;Nikkilä J;Marchesi JR;Smidt H;de Vos WM;Ross RP;O'Toole PW
通讯作者：
O'Toole PW
影响因子：
56.9
作者：
Gill, Steven R.;Pop, Mihai;Nelson, Karen E.
通讯作者：
Nelson, Karen E.
DOI：
10.1073/pnas.0504978102
发表时间：
2005-08-02
影响因子：
11.1
作者：
Ley, RE;Bäckhed, F;Gordon, JI
通讯作者：
Gordon, JI