A novel ultra high-throughput 16S rRNA gene amplicon sequencing library preparation method for the Illumina HiSeq platform.
A novel ultra high-throughput 16S rRNA gene amplicon sequencing library preparation method for the Illumina HiSeq platform.
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DOI：
10.1186/s40168-017-0279-1
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发表时间：
2017-07-06
期刊：
影响因子：
15.5
通讯作者：
Sundaram AYM
中科院分区：
文献类型：
--
作者：
de Muinck EJ;Trosvik P;Gilfillan GD;Hov JR;Sundaram AYM
测序技术和生物信息学的进步使微生物群落的分析几乎成为常规。尽管如此，仍然需要改进用于收集此类数据的技术，包括在降低成本的同时提高吞吐量和对技术进行基准测试，以便更好地表征潜在的偏见来源。与现有方法相比，我们提出了一种三指数扩增子测序策略，以显着降低的成本和更短的时间框架对大量样品进行测序。该设计采用两阶段PCR协议，将三个条形码纳入每个样本，并可能添加第四个索引。它还包括异质性间隔器，以克服在Illumina平台上测序扩增子时面临的低复杂性问题。通过对模拟群落的分析，对文库制备方法进行了广泛的基准测试，以评估样本索引、PCR循环次数和模板浓度带来的偏差。我们通过标准化环境样本的重新测序进一步评估了该方法。最后，我们在一小群健康成年人的粪便样本上评估了我们的方案，在现实的实验环境中证明了良好的性能。样本间差异主要与DNA提取等批效应有关，而样本索引也是偏差的重要来源。PCR循环数强烈影响嵌合体的形成，并影响高GC含量物种的相对丰度估计。使用Illumina HiSeq和MiSeq平台对文库进行测序，以证明该协议具有高度可扩展性，可以以非常低的成本对数千个样本进行测序。在这里，我们提供了迄今为止16S rRNA基因扩增子测序方法固有的性能和偏差的最全面的研究。三重索引极大地减少了文库制备所需的长定制DNA寡核苷酸的数量，而包含可变长度异质性间隔器则最大限度地减少了对PhiX插入的需求。这种设计显著降低了高复用扩增子测序的成本。我们所描述的偏见突出了高度标准化协议的必要性。令人欣慰的是，我们发现生物信号是一个比各种偏见来源更强大的结构性因素。本文的在线版本（doi:10.1186/s40168-017-0279-1）包含补充材料，可供授权用户使用。
Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized. We present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms. The library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost. Here, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias. The online version of this article (doi:10.1186/s40168-017-0279-1) contains supplementary material, which is available to authorized users.
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DOI：
10.1038/ismej.2012.8
发表时间：
2012-08
期刊：
The ISME journal
影响因子：
--
作者：
通讯作者：
--
影响因子：
15.5
作者：
Fadrosh DW;Ma B;Gajer P;Sengamalay N;Ott S;Brotman RM;Ravel J
通讯作者：
Ravel J
DOI：
10.1038/ismej.2011.208
发表时间：
2012-07
期刊：
The ISME journal
影响因子：
--
作者：
通讯作者：
--
影响因子：
4.4
作者：
Kozich, James J.;Westcott, Sarah L.;Schloss, Patrick D.
通讯作者：
Schloss, Patrick D.
PCR在焦磷酸测序数据集中偏离细菌和古细菌群落结构。
影响因子：
3.7
作者：
Pinto AJ;Raskin L
通讯作者：
Raskin L