Abstract
High-throughput sequencing is a powerful tool used for bivalve symbiosis research, but the largest barrier is the contamination of host DNA. In this work, we assessed the host DNA reduction efficiency, microbial community structure, and microbial diversity of four different sample pre-treatment and DNA extraction methods employed in bivalve gill tissue samples. Metagenomic sequencing showed the average proportions of reads belonging to microorganisms retrieved using PowerSoil DNA extraction kit, pre-treatment with differential centrifugation, pre-treatment with filtration, and HostZERO Microbial DNA kit samples were 2.3 ± 0.6%, 2.5 ± 0.2%, 4.7 ± 1.6%, and 42.6 ± 6.8%, respectively. The microbial DNA was effectively enriched with HostZERO Microbial DNA kit. The microbial communities revealed by amplicon sequencing of the 16S rRNA gene showed the taxonomic biases by using four different pre-treatment and DNA extraction methods. The species diversities of DNA samples extracted with the PowerSoil DNA extraction kit were similar, while lower than DNA samples extracted with HostZERO Microbial DNA kit. The results of this study emphasized the bias of these common methods in bivalve symbionts research and will be helpful to choose a fit-for-purpose microbial enrichment strategy in future research on bivalves or other microbe–invertebrate symbioses.
Highlights
Published: 8 February 2022Bivalves are widely distributed from freshwater to marine environments and from coastal areas to the deep sea [1,2]
The comparison of the bacterial communities obtained using these four methods further showed that the communities obtained using the HostZERO microbial DNA kit formed a separate cluster and bacterial communities retrieved using PowerSoil DNA extraction kit, pre-treatment with differential centrifugation, and pre-treatment with filtration were similar (Figure 5a)
The results showed that the pre-treatment method of filtration did not significantly increase the proportion of microbial-derived reads in the metagenomic libraries
Summary
Bivalves are widely distributed from freshwater to marine environments and from coastal areas to the deep sea [1,2]. At present, obtaining bivalve symbiotic microbial DNA includes two major approaches: one is extraction of genomic DNA from bivalve samples directly using CTAB method [17,30] or commercial kits, such as PowerSoil DNA extraction kit, which was commonly used in previous studies [31,32]. These methods might result in the quantity of host DNA deeply exceeding microbial DNA, limiting the sensitivity of nucleic acidbased microbial diagnostic systems [33–35]. In order to identify preferred methods to enrich microbial DNA from bivalve samples and evaluate the bias introduced by different methods, we chose Perna canaliculus gill tissue as material because it is easy to acquire from markets and the result obtained from it is representative. The host DNA depletion efficiency was evaluated, and the bias of these methods was tested by comparing the retrieved microbial communities
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