High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

Minich, Jeremiah J.; Humphrey, Greg; Benitez, Rodolfo A. S.; Sanders, Jon G.; Swafford, Austin D.; Allen, Eric E.; Knight, Rob

doi:10.1128/msystems.00166-18

Cited by 67 publications

(58 citation statements)

References 23 publications

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“…A total of 21 positive controls of a microbial isolate (replicates of 10 fold serial dilutions) were processed alongside the samples and then used to determine sample success rate by calculating the sample exclusion criteria based on read counts described in the Katharoseq method (27). Samples were processed in triplicate 5 ul PCR reactions (34)[PMID: 30417111] using the 16S v4 515/806 primers (35, 36) and then pooled at equal volume according to Katharoseq (27). The final amplicon pool was processed using the Qiagen PCR cleanup kit following EMP protocols and sequenced on a MiSeq 2×250 bp run (37).…”

Section: Methodsmentioning

confidence: 99%

Microbial ecology of Atlantic salmon, Salmo salar, hatcheries: impacts of the built environment on fish mucosal microbiota

Minich

Jantawongsri

Johnston³

et al. 2019

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ABSTRACTSuccessful rearing of fish in hatcheries is critical for conservation, recreational fishing, and commercial fishing through wild stock enhancements, and aquaculture production. Flow through (FT) hatcheries require more water than Recirculating-Aquaculture-Systems (RAS) which enable up to 99% of water to be recycled thus significantly reducing environmental impacts. Here, we evaluated the biological and physical microbiome interactions of the built environment of a hatchery from three Atl salmon hatcheries (RAS n=2, FT n=1). Six juvenile fish were sampled from tanks in each of the hatcheries for a total of 60 fish across 10 tanks. Water and tank side biofilm samples were collected from each of the tanks along with three salmon body sites (gill, skin, and digesta) to assess mucosal microbiota using 16S rRNA sequencing. The water and tank biofilm had more microbial richness than fish mucus while skin and digesta from RAS fish had 2× the richness of FT fish. Body sites each had unique microbial communities (P<0.001) and were influenced by the various hatchery systems (P<0.001) with RAS systems more similar. Water and especially tank biofilm richness was positively correlated with skin and digesta richness. Strikingly, the gill, skin and digesta communities were more similar to the origin tank biofilm vs. all other experimental tanks suggesting that the tank biofilm has a direct influence on fish-associated microbial communities. The results from this study provide evidence for a link between the tank microbiome and the fish microbiome with the skin microbiome as an important intermediate.IMPORTANCEAtlantic salmon, Salmo salar, is the most farmed marine fish worldwide with an annual production of 2,248 million metric tonnes in 2016. Salmon hatcheries are increasingly changing from flow through towards RAS design to accommodate more control over production along with improved environmental sustainability due to lower impacts on water consumption. To date, microbiome studies on hatcheries have focused either on the fish mucosal microbiota or the built environment microbiota, but have not combined the two to understand interactions. Our study evaluates how water and tank biofilm microbiota influences fish microbiota across three mucosal environments (gill, skin, and digesta). Results from this study highlight how the built environment is a unique source of microbes to colonize fish mucus and furthermore how this can influence the fish health. Further studies can use this knowledge to engineer built environments to modulate fish microbiota for a beneficial phenotype.

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Section: Methodsmentioning

confidence: 99%

Microbial ecology of Atlantic salmon, Salmo salar, hatcheries: impacts of the built environment on fish mucosal microbiota

Minich

Jantawongsri

Johnston³

et al. 2019

Preprint

Self Cite

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“…Massively high-throughput sequencing has enabled fundamental changes to the study of microbial ecology. Increased throughput and sequencing depth has empowered researchers to utilize multiplexing to increase sample sizes to thousands per study [1–6]. However, new ways of knowing require new understanding of potential flaws and confounds.…”

Section: Introductionmentioning

confidence: 99%

Quantifying and understanding well-to-well contamination in microbiome research

Minich

Sanders

Almasi‐Hashiani

et al. 2019

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Microbial sequences inferred as belonging to one sample may not have originated from that sample. Such contamination may arise from laboratory or reagent sources or from physical exchange between samples. This study seeks to rigorously assess the behavior of this often-neglected between-sample contamination. Using unique bacteria each assigned a particular well in a plate, we assess the frequency at which sequences from each source appears in other wells. We evaluate the effects of different DNA extraction methods performed in two labs using a consistent plate layout including blanks, low biomass, and high biomass samples. Well-to-well contamination occurred primarily during DNA extraction, and to a lesser extent in library preparation, while barcode leakage was negligible. Labs differed in the levels of contamination. DNA extraction methods differed in their occurrences and levels of well-to-well contamination, with robotic methods having more well-to-well contamination while manual methods having higher background contaminants. Well-to-well contamination was observed to occur primarily in neighboring samples, with rare events up to 10 wells apart. The effect of well-to-well was greatest in samples with lower biomass, and negatively impacted metrics of alpha and beta diversity. Our work emphasizes that sample contamination is a combination of crosstalk from nearby wells and background contaminants. To reduce well-to-well effects, samples should be randomized across plates, and samples of similar biomass processed together. Researchers should evaluate well-to-well contamination in study design and avoid removal of taxa or OTUs appearing in negative controls, as many will be microbes from other samples rather than reagent contaminants.ImportanceMicrobiome research has uncovered magnificent biological and chemical stories across nearly all areas of life science, at times creating controversy when findings reveal fantastic descriptions of microbes living and even thriving in once thought to be sterile environments. Scientists have refuted many of these claims because of contamination, which has led to robust requirements including use of controls for validating accurate portrayals of microbial communities. In this study, we describe a previously undocumented form of contamination, well-to-well contamination and show that contamination primarily occurs during DNA extraction rather than PCR, is highest in plate-based methods as compared to single tube extraction, and occurs in higher frequency in low biomass samples. This finding has profound importance on the field as many current techniques to ‘decontaminate’ a dataset simply relies on an assumption that microbial reads found in blanks are contaminants from ‘outside’ namely the reagents or consumables.

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“…For the general sample types tested here, we recommend using single PCRs rather than triplicate PCRs. Combined with other technical improvements in miniaturizing PCR reactions [13], this change in protocol will substantially reduce the cost and complexity of amplicon studies.…”

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confidence: 99%

Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

et al. 2019

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Conventional wisdom holds that PCR amplification for sequencing should employ pooled replicate reactions to reduce bias due to jackpot effects and chimera formation. However, modern amplicon data analysis employs methods that may be less sensitive to such artifacts. Here we directly compare results from single versus triplicate reactions for 16S amplicon sequencing and find no significant impact of adopting a less labor-intensive single-reaction protocol.

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High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

Cited by 67 publications

References 23 publications

Microbial ecology of Atlantic salmon, Salmo salar, hatcheries: impacts of the built environment on fish mucosal microbiota

Microbial ecology of Atlantic salmon, Salmo salar, hatcheries: impacts of the built environment on fish mucosal microbiota

Quantifying and understanding well-to-well contamination in microbiome research

Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

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