Microvolume DNA extraction methods for microscale amplicon and metagenomic studies

Bramucci, Anna R.; Focardi, Amaranta; Hugenholtz, Philip; Tyson, Gene W.; Seymour, Justin R.

doi:10.1038/s43705-021-00079-z

Cited by 18 publications

(13 citation statements)

References 24 publications

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“…When compared to the concurrent Niskin bottle samples, the sampler captured a near-identical community for both bacteria and phytoplankton at all stations. This is despite differences in the protocol such as volume filtered and temporal resolution, aligning with prior studies, which have shown similar results 42,43 . A recent study performed using the 3G ESP also demonstrated that results were equivalent between autonomous and manual sampling 44 .…”

Section: Discussionsupporting

confidence: 89%

Compact and automated eDNA sampler for in situ monitoring of marine environments

Hendricks

Mackie²,

Luy³

et al. 2023

Sci Rep

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Using environmental DNA (eDNA) to monitor biodiversity in aquatic environments is becoming an efficient and cost-effective alternative to other methods such as visual and acoustic identification. Until recently, eDNA sampling was accomplished primarily through manual sampling methods; however, with technological advances, automated samplers are being developed to make sampling easier and more accessible. This paper describes a new eDNA sampler capable of self-cleaning and multi-sample capture and preservation, all within a single unit capable of being deployed by a single person. The first in-field test of this sampler took place in the Bedford Basin, Nova Scotia, Canada alongside parallel samples taken using the typical Niskin bottle collection and post-collection filtration method. Both methods were able to capture the same aquatic microbial community and counts of representative DNA sequences were well correlated between methods with R$$^{2}$$ 2 values ranging from 0.71–0.93. The two collection methods returned the same top 10 families in near identical relative abundance, demonstrating that the sampler was able to capture the same community composition of common microbes as the Niskin. The presented eDNA sampler provides a robust alternative to manual sampling methods, is amenable to autonomous vehicle payload constraints, and will facilitate persistent monitoring of remote and inaccessible sites.

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Section: Discussionsupporting

confidence: 89%

Compact and automated eDNA sampler for in situ monitoring of marine environments

Hendricks

Mackie²,

Luy³

et al. 2023

Sci Rep

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“…By collectively analyzing the concentrations of tetA , ermB , qnrS and bla CTX-M obtained from AWW2 to AWW4, EP1, EP2 and EP5 using 0.2/0.5 mL sample volume with no pre-spin step yielded significantly greater ARG concentrations than in EP3 and EP9 using 1.0 mL and EP6 using 0.5 mL sample volume. The superior performance of small sample volumes for nucleic acid extraction has also been observed in previous studies [ 7 , 22 , 28 ]. On the one hand, employing small sample volume may minimize the interference of toilet paper during the extraction process, which may introduce PCR inhibitors and compromise amplification efficiency [ 11 ].…”

Section: Discussionsupporting

confidence: 76%

Assessment of nucleic acid extraction protocols for antibiotic resistance genes (ARGs) quantification in aircraft wastewater

Smith,

Liu,

Simpson

et al. 2024

Hum Genomics

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This study evaluated ten nucleic acid extraction protocols (EP1 to EP10) for measuring five endogenous antibiotic resistance genes (ARGs) in four aircraft wastewater samples (AWW1 to AWW4). The targeted ARGs, including blaCTX-M, blaNDM-1, ermB, qnrS, and tetA, encompassed highly and minimally abundant ARGs. TetA and ermB were consistently detected across four aircraft wastewater samples using the DNeasy Blood and Tissue Kit and the AllPrep PowerViral DNA/RNA kit. QnrS displayed high detection rates with specific extraction protocols and aliquot volumes. Concentrations of ARGs varied across aircraft wastewater samples, with differing extraction protocols influencing quantitative results. The concentrations of tetA, ermB, and qnrS in AWW1 were distinct, while AWW2 to AWW4 exhibited a broader range for tetA, ermB, qnrS, blaCTX-M, and blaNDM-1. EP1 consistently produced the highest concentrations for several ARGs. Collective data analysis revealed varying ARG concentrations across the ten extraction protocols, suggesting the importance of careful extraction protocol selection in ARG monitoring in aircraft wastewater samples. Based on the results, we suggest that a small sample volume (as low as 0.2 mL) may be sufficient for ARG characterization in aircraft wastewater samples. The findings also emphasize the need for considering toilet paper removal without compromising nucleic acid extraction efficiency. The study highlights promising prospects for aircraft wastewater monitoring of ARGs, calling for further investigation into the import and spread of unique ARGs through transport hubs.

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“…Ideally, researchers should aim to collect enough cells so that amplification is not necessary. In these cases, DNA can be extracted with low biomass-input methods (Bramucci et al 2021 ). When this is not possible, a potential alternative to MDA is primary template-directed amplification (PTA) (Gonzalez-Pena et al 2021 ).…”

Section: Resultsmentioning

confidence: 99%

A targeted approach to enrich host-associated bacteria for metagenomic sequencing

Dungan,

Tandon,

Jameson

et al. 2023

FEMS Microbes

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Multicellular eukaryotic organisms are hosts to communities of bacteria that reside on or inside their tissues. Often the eukaryotic members of the system contribute to high proportions of metagenomic sequencing reads, making it challenging to achieve sufficient sequencing depth to evaluate bacterial ecology. Stony corals are one such complex community; however, separation of bacterial from eukaryotic (primarily coral and algal symbiont) cells has so far not been successful. Using a combination of hybridization chain reaction fluorescence in situ hybridization and fluorescence activated cell sorting (HCR-FISH + FACS), we sorted two populations of bacteria from five genotypes of the coral Acropora loripes, targeting 1) Endozoicomonas spp, and 2) all other bacteria. NovaSeq sequencing resulted in 67-91 M reads per sample, 55–90% of which were identified as bacterial. Most reads were taxonomically assigned to the key coral-associated family, Endozoicomonadaceae, with Vibrionaceae also abundant. Endozoicomonadaceae were 5x more abundant in the ‘Endozoicomonas’ population, highlighting the success of the dual-labelling approach. This method effectively enriched coral samples for bacteria with <1% contamination from host and algal symbionts. The application of this method will allow researchers to decipher the functional potential of coral-associated bacteria. This method can also be adapted to accommodate other host-associated communities.

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Microvolume DNA extraction methods for microscale amplicon and metagenomic studies

Cited by 18 publications

References 24 publications

Compact and automated eDNA sampler for in situ monitoring of marine environments

Compact and automated eDNA sampler for in situ monitoring of marine environments

Assessment of nucleic acid extraction protocols for antibiotic resistance genes (ARGs) quantification in aircraft wastewater

A targeted approach to enrich host-associated bacteria for metagenomic sequencing

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