[Biases on community structure during DNA extraction of shrimp intestinal microbiota revealed by high-throughput sequencing].
[Biases on community structure during DNA extraction of shrimp intestinal microbiota revealed by high-throughput sequencing].
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发表时间：
2016-01
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通讯作者：
C. Wen;Yaoyao He;M. Xue;Huafang Liang;Junde Dong
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作者：
C. Wen;Yaoyao He;M. Xue;Huafang Liang;Junde Dong
高通量测序技术越来越多地应用于包括虾在内的水生动物的肠道微生物群。然而，目前缺乏从虾肠道菌群中提取DNA的标准方法或试剂盒，并且对基于高通量测序的DNA提取的有效性和偏倚知之甚少。本研究旨在通过高通量测序技术研究不同DNA提取试剂盒对凡纳滨对虾肠道菌群结构的影响，以更好地了解健康凡纳滨对虾肠道菌群植物群的结构和组成。方法提取肠道菌群总DNA。使用三种设计用于从细菌、粪便和组织中提取DNA的商业试剂盒（Omega，USA）对vannamei进行DNA提取。基于NanoDrop的260/280 nm的吸光度比评价DNA质量，同时使用PicoGreen定量DNA浓度。采用Illumina MiSeq高通量测序技术对16 S rDNA V4区进行PCR扩增，检测肠道细菌群落结构。结果细菌试剂盒（SIB）的DNA产量和纯度均上级粪便试剂盒（SIS），而组织试剂盒（SIT）的DNA量太少，不能有效扩增。从SIB和SIS样品获得的平均序列读数分别为52151 ± 5085和55296 ± 5147。在相同的46800个读段的深度上进行重定位后，SIS样本的操作分类单位（OTU）数和Shannon多样性指数显著高于SIB样本。与此相反，OTU在SIB重复之间的重复性高于SIS重复之间。SIS和SIB样品的优势门是相同的，包括变形菌门、厚壁菌门、拟杆菌门、浮游菌门、放线菌门和蓝细菌门。然而，几乎所有的优势类群在不同的分类水平的相对丰度在这两个样品之间有很大的差异。结论DNA提取对对虾肠道菌群结构有明显的影响。而健康L.本研究中的凡纳滨对虾主要由发光杆菌属、乳球菌属、弧菌属、弧菌属以及其他三个未分类的类群组成。
OBJECTIVES High-throughput sequencing technology is increasingly applied in intestinal microbiota of aquatic animals including shrimp. However, there is a lack of standard method or kit for DNA isolation from shrimp intestinal microbiota, and little is known about the effectiveness and biases regarding DNA extraction based on high-throughput sequencing. The aim of this study was to study the biases of different DNA extraction kits on community structure of shrimp intestinal microbiota through high-throughput sequencing, and to better understand the structure and composition of bacterial flora associated with healthy Litopenaeus vannamei. METHODS We extracted the total DNA of intestinal microbiota from L. vannamei with three commercial kits designed for DNA extraction from bacteria, stool and tissue (Omega, USA). DNA quality was evaluated based on the absorbance ratios of 260/280 nm by NanoDrop, while DNA concentration was quantified using PicoGreen. Then Illumina MiSeq high-throughput sequencing was used to examine the intestinal bacterial communities following PCR amplification of 16S rDNA V4 region. RESULTS The yield and purity of the DNA from the Bacterial Kit (SIB) were superior to those from the Stool Kit (SIS), whereas the DNA from Tissue Kit (SIT) presented too small amount to be amplified efficiently. The average sequence reads obtained from SIB and SIS samples were 52151 ± 5085 and 55296 ± 5147 respectively. After resampling at the same depth of 46800 reads, the operational taxonomic unit (OTU) number and Shannon diversity index of SIS samples were significantly higher than those of SIB samples. By contrast, the reproducibility of OTU among SIB replicates was higher than that among SIS replicates. The dominant phyla of SIS and SIB samples were identical, including Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, Actinobacteria, and Cyanobacteria. However, the relative abundances of almost all the dominant groups at various taxonomic levels differed greatly between these two samples. CONCLUSION Significant biases on community structure of shrimp intestinal microbiota were detected which originated from DNA extraction. And the core microbiota of the healthy L. vannamei in this study was mainly composed of genera Photobacterium, Lactococcus, Aliivibrio, Vibrio, as well as three other unclassified groups.