Abstract
This study examined the technical bias associated with different DNA extraction methods used in microbiome research. Three methods were used to extract genomic DNA from the same intestinal microbiota sample that was taken from the koi carp Cyprinus carpio var. koi, after which their microbial diversity and community structure were investigated on the basis of a 16S rDNA high‐throughput sequencing analysis. Biased results were observed in relation to the number of reads, alpha diversity indexes and taxonomic composition among the three DNA extraction protocols. A total of 1,381 OTUs from the intestinal bacteria were obtained, with 852, 759, and 698 OTUs acquired, using the Lysozyme and Ultrasonic Lysis method, Zirmil‐beating Cell Disruption method, and a QIAamp Fast DNA Stool Mini Kit, respectively. Additionally, 336 OTUs were commonly acquired, using the three methods. The results showed that the alpha diversity indexes (Rarefaction, Shannon, and Chao1) of the community that were determined using the Lysozyme and Ultrasonic Lysis method were higher than those obtained with the Zirmil‐beating Cell Disruption method, while the Zirmil method results were higher than those measured, using the QIAamp Fast DNA Stool Mini Kit. Moreover, all the major phyla (ratio>1%) could be identified with all three DNA extraction methods, but the phyla present at a lower abundance (ratio <1%) could not. Similar findings were observed at the genus level. Taken together, these findings indicated that the bias observed in the results about the community structure occurred primarily in OTUs with a lower abundance. The results of this study demonstrate that possible bias exists in community analyses, and researchers should therefore be conservative when drawing conclusions about community structures based on the currently available DNA extraction methods.
Highlights
| INTRODUCTIONNew technologies based on meta-genomics/high-throughput sequencing have been developed and successfully applied to analyzing the complex bacterial ecosystem of the gut
Increasing evidence shows that 16S ribosomal DNA (rDNA) sequencing approaches can be used to identify bacteria rapidly because they can overcome the limitations of culture- based bacterial detection methods
TA B L E 1 Alpha diversity index, number of operational taxonomic units (OTUs) and filtered reads from DNA samples extracted with the CLU, Zirmil- beating Cell Disruption method (ZBC) or QIA methods
Summary
New technologies based on meta-genomics/high-throughput sequencing have been developed and successfully applied to analyzing the complex bacterial ecosystem of the gut. These new analytical approaches usually involve DNA extraction from stool samples or biopsies and the amplification of 16S ribosomal DNA (rDNA) followed by high- throughput sequencing. Several protocols for extracting DNA from fish intestinal microflora have been described, including physical and chemical methods. This study examined the bias in results that were obtained using different extraction methods during microbiome research based on 16S rDNA high-throughput sequencing analyses. Three methods were used to extract the genomic DNA from the same sample of intestinal microbiota from the koi carp, Cyprinus carpio var. A protocol was modified from the lysozyme method developed by our laboratory and named the Combination of Lysozyme and Ultrasonic Lysis method (CLU); the Zirmil- beating Cell Disruption method (ZBC) referring to the research of Zoetendal et al (2006) and a QIAamp Fast DNA Stool Mini Kit (QIA, Qiagen, Hilden, Germany), a common commercial kit, were used
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