DNA Enrichment Methods for Microbial Symbionts in Marine Bivalves

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.

Novel Virus Identification through Metagenomics: A Systematic Review

“…It has been shown that the use of pre-extraction enrichment methods can introduce bias in the identification of microbial species present in the sample [283]. For example, filtration can reduce the abundance of bacterial [284] or viral species [285,286], depending on pore size dimension. Similarly, enrichment based on low-force centrifugation can induce depletion of large viruses [285].…”

A Comprehensive Review for the Surveillance of Human Pathogenic Microorganisms in Shellfish

“…Some kits are designed for samples containing methylated host DNA, which are not supposed to be used for invertebrate genome depletion due to sparse methylation with a mosaic pattern in marine invertebrates genomes [ 187 , 188 ], while others perform the differential lysis of host vs bacterial cells [ 189 , 190 ]. Some have been successfully applied for bivalve symbiont enrichment [ 191 ]. Finally, osmotic lysis combined with PMA (lyPMA) seems to be a promising and cost-effective approach [ 192 ].…”

Alleviating Salivary Microbiome Dysbiosis Associated with Dental Malocclusion Utilizing Low-level Laser Therapy

“…These methods contribute to our understanding of microbial symbioses, mucocutaneous niches, and the overall microbiome. Future research should explore the potential of these methods in whole-genome sequencing and probe design for metagenomes (26)(27)(28)(29) . In analyzing oral microbiome diversity after LLLT therapy in dental malocclusion subjects, techniques like DNA sequencing and microbial enrichment can provide comprehensive insights into the oral microbiome's response to low-level laser treatment.…”