A new Illumina MiSeq high‐throughput sequencing‐based method for evaluating the composition of the Bacteroides community in the intestine using the rpsD gene sequence,Microbial Biotechnology

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A new Illumina MiSeq high‐throughput sequencing‐based method for evaluating the composition of the Bacteroides community in the intestine using the rpsD gene sequence
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2020-08-11 , DOI: 10.1111/1751-7915.13651
Chen Wang _{1,

2} , Saisai Feng _{1,

2} , Yue Xiao _{1,

2} , Mingluo Pan _{1,

2} , Jianxin Zhao _{1,

2} , Hao Zhang _{1,

2,

3,

4,

5} , Qixiao Zhai _{1,

2,

6} , Wei Chen _{1,

2,

3,

7}

Affiliation

Bacteroides is a bacterial genus that is known to closely interact with the host. The potential role of this genus is associated with its ecological status and distribution in the intestine. However, the current 16S V3–V4 region sequencing method can only detect the abundance of this genus, revealing a need for a novel sequencing method that can elucidate the composition of Bacteroides in the human gut microbiota. In this study, a core gene, rpsD, was selected as a template for the design of a Bacteroides‐specific primer set. We used this primer set to develop a novel assay based on the Illumina MiSeq sequencing platform that enabled an accurate assessment of the Bacteroides compositions in complex samples. Known amounts of genomic DNA from 10 Bacteroides species were mixed with a complex sample and used to evaluate the performance and detection limit of our assay. The results were highly consistent with those of direct sequencing with a low Bacteroides DNA detection threshold (0.01 ng), supporting the reliability of our assay. In addition, the assay could detect all the known Bacteroides species within the faecal sample. In summary, we provide a sensitive and specific approach to determining the Bacteroides species in complex samples.

中文翻译：

一种基于 Illumina MiSeq 高通量测序的新方法，使用 rpsD 基因序列评估肠道中拟杆菌群落的组成

拟杆菌是已知与宿主密切相互作用的细菌属。该属的潜在作用与其在肠道中的生态状况和分布有关。然而，目前的 16S V3-V4 区测序方法只能检测该属的丰度，这表明需要一种新的测序方法来阐明人类肠道微生物群中拟杆菌的组成。在本研究中，选择核心基因rpsD作为设计拟杆菌属特异性引物组的模板。我们使用该引物组开发了一种基于 Illumina MiSeq 测序平台的新型检测方法，能够准确评估复杂样品中的拟杆菌成分。将来自 10 个拟杆菌属物种的已知量的基因组 DNA 与复杂样品混合，用于评估我们测定的性能和检测限。结果与低拟杆菌DNA 检测阈值（0.01 ng）的直接测序结果高度一致，支持了我们测定的可靠性。此外，该检测还可以检测粪便样本中所有已知的拟杆菌属。总之，我们提供了一种灵敏且特异的方法来确定复杂样品中的拟杆菌属。

更新日期：2020-08-11

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