Combined nanopore adaptive sequencing and enzyme-based host depletion efficiently enriched microbial sequences and identified missing respiratory pathogens

Gan, Mingyu; Wu, Bingbing; Yan, Gangfeng; Li, Gang; Sun, Li; Li, Guoping; Zhou, Wenhao

doi:10.1186/s12864-021-08023-0

Cited by 34 publications

(34 citation statements)

References 31 publications

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“…Compared with the PowerSoil DNA extraction kit, the HostZERO Microbial DNA kit could significantly reduce the proportion of host DNA as well as increase the diversity of both bacteria and archaea detected. This result was consistent with previous studies on respiratory tract samples [56]. As the proportion of host DNA was reduced, some low-abundance microorganisms could be detected and the microbial community showed higher species diversity [57].…”

Section: Discussionsupporting

confidence: 92%

DNA Enrichment Methods for Microbial Symbionts in Marine Bivalves

et al. 2022

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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.

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Section: Discussionsupporting

confidence: 92%

DNA Enrichment Methods for Microbial Symbionts in Marine Bivalves

et al. 2022

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“…ONT’s adaptive sampling method demonstrated in our work a 1.7 fold increase in sequencing depth for samples with high human DNA contamination, which increases sensitivity of taxonomic profiling by providing more sequencing data 2 . Moreover, adaptive sampling can be used in addition to wet lab procedures to increase sensitivity further 37 . This makes molecular monitoring of human reservoirs with low microbial concentrations and high host DNA loads (e.g., nasal swabs, sputum, or skin swabs) more feasible.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

Marquet

Zöllkau

Pastuschek

et al. 2022

Sci Rep

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Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective ‘human host depletion’ resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.

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“…Nanopore adaptive sequencing is a relatively new bioinformatic pipeline (Payne et al 2021), thus comparisons to traditional nanopore sequencing runs are essential. The analyses presented herein and those of others (Gan et al, 2021;Kipp et al, 2021;Martin et al, 2021;Payne et al, 2021;Wanner et al, 2021) (Hlaing et al, 2009);…”

Section: Nas Performance Versus Control Sequencingmentioning

confidence: 52%

Nanopore adaptive sampling for mitogenome sequencing and bloodmeal identification in hematophagous insects

Kipp

Lindsey

Blanco

et al. 2021

Preprint

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Blood-feeding insects are important vectors for an array of zoonotic pathogens. Despite significant research focused on well-documented insect vectors of One Health importance, resources for molecular species identification of a large number of hematophagous arthropods are limited. Advancements in next-generation sequencing technologies provide opportunities for targeting mitochondrial genomes of blood-feeding insects, as well as their bloodmeal hosts. This dual approach holds great promise for elucidating complex disease transmission pathways and enhancing the molecular resources for the identification of cryptic insect species. To this end, we leveraged the newly developed Oxford Nanopore Adaptive Sampling (NAS) pipeline to dually sequence the mitogenomes of hematophagous insects and their bloodmeals. Using NAS, we sequenced the entire mitogenomes of Aedes vexans, Culex restuans, Culex territans, and Chrysops niger and successfully identified bloodmeal hosts of Chrysops niger, Culex restuans, and Aedes trivittatus. We show that NAS has the utility to simultaneously molecularly identify blood-feeding insects and characterize disease transmission pathways through bloodmeal host identification. Moreover, our data indicate NAS can facilitate a wide array of molecular systematic studies through novel ‘phylogenetic capture’ methods. We conclude the NAS approach has great potential for informing global One Health initiatives centered on the mitigation of vector-borne disease through dual vector and bloodmeal identification.

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Combined nanopore adaptive sequencing and enzyme-based host depletion efficiently enriched microbial sequences and identified missing respiratory pathogens

Cited by 34 publications

References 31 publications

DNA Enrichment Methods for Microbial Symbionts in Marine Bivalves

DNA Enrichment Methods for Microbial Symbionts in Marine Bivalves

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

Nanopore adaptive sampling for mitogenome sequencing and bloodmeal identification in hematophagous insects

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