Biases on community structure during DNA extraction of shrimp intestinal microbiota revealed by high-throughput sequencing

Abstract:

[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.