Metagenomics as a Tool To Monitor Reclaimed-Water Quality

Hong, Pei‐Ying; Mantilla‐Calderon, David; Wang, Changzhi

doi:10.1128/aem.00724-20

Cited by 17 publications

(7 citation statements)

References 73 publications

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“…Methods designed for effectively concentrating and purifying DNA viral genomes may not be as effective for RNA viruses. The necessary reverse transcription step to form cDNA and the need to deplete host RNA, for example, add unique challenges in preparing unbiased RNA viral communities from sequencing ( Hong, Mantilla-Calderon & Wang, 2020 ). The unknown relative abundance of RNA viruses in water samples further complicates RNA viral library preparation.…”

Section: Discussionmentioning

confidence: 99%

Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types

Langenfeld

Chin

Roy

et al. 2021

PeerJ

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Viral metagenomes (viromes) are a valuable untargeted tool for studying viral diversity and the central roles viruses play in host disease, ecology, and evolution. Establishing effective methods to concentrate and purify viral genomes prior to sequencing is essential for high quality viromes. Using virus spike-and-recovery experiments, we stepwise compared two common approaches for virus concentration, ultrafiltration and iron chloride flocculation, across diverse matrices: wastewater influent, wastewater secondary effluent, river water, and seawater. Viral DNA was purified by removing cellular DNA via chloroform cell lysis, filtration, and enzymatic degradation of extra-viral DNA. We found that viral genomes were concentrated 1-2 orders of magnitude more with ultrafiltration than iron chloride flocculation for all matrices and resulted in higher quality DNA suitable for amplification-free and long-read sequencing. Given its widespread use and utility as an inexpensive field method for virome sampling, we nonetheless sought to optimize iron flocculation. We found viruses were best concentrated in seawater with five-fold higher iron concentrations than the standard used, inhibition of DNase activity reduced purification effectiveness, and five-fold more iron was needed to flocculate viruses from freshwater than seawater—critical knowledge for those seeking to apply this broadly used method to freshwater virome samples. Overall, our results demonstrated that ultrafiltration and purification performed better than iron chloride flocculation and purification in the tested matrices. Given that the method performance depended on the solids content and salinity of the samples, we suggest spike-and-recovery experiments be applied when concentrating and purifying sample types that diverge from those tested here.

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Section: Discussionmentioning

confidence: 99%

Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types

Langenfeld

Chin

Roy

et al. 2021

PeerJ

View full text Add to dashboard Cite

show abstract

“…Methods designed for effectively concentrating and purifying DNA viral genomes may not be as effective for RNA viruses. The necessary reverse transcription step to form cDNA and the need to deplete host RNA, for example, add unique challenges in preparing unbiased RNA viral communities from sequencing (72). The unknown relative abundance of RNA viruses in water samples further complicates RNA viral library preparation.…”

Section: Limitations Of Phage Spike-and-recovery Experiments Selected Matrices and Focus Onmentioning

confidence: 99%

Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types

Langenfeld

Chin

Wigginton

et al. 2020

Preprint

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Viral metagenomes (viromes) are a valuable untargeted tool for studying viral diversity and the central roles viruses play in host disease, ecology, and evolution. Establishing effective methods to concentrate and purify viral genomes prior to sequencing is essential for high quality viromes. Using virus spike-and-recovery experiments, we stepwise compared two common approaches for virus concentration, ultrafiltration and iron chloride flocculation, across diverse matrices: wastewater influent, wastewater secondary effluent, river water, and seawater. Viral DNA was purified by removing cellular DNA via chloroform cell lysis, filtration, and enzymatic degradation of extra-viral DNA. We found that viral genomes were concentrated 1-2 orders of magnitude more with ultrafiltration than iron chloride flocculation for all matrices and resulted in higher quality DNA suitable for amplification-free and long-read sequencing. Given its widespread use and utility as an inexpensive field method for virome sampling, we nonetheless sought to optimize iron flocculation. We found viruses were best concentrated in seawater with 25-fold higher iron concentrations than the standard used, inhibition of DNase activity reduced purification effectiveness, and five-fold more iron was needed to flocculate viruses from freshwater than seawater-critical knowledge for those seeking to apply this broadly used method to freshwater virome samples. Overall, our results demonstrated that ultrafiltration and purification performed better than iron chloride flocculation and purification in the tested matrices. Given that the method performance depended on the solids content and salinity of the samples, we suggest spike-and-recovery experiments be applied when concentrating and purifying sample types that diverge from those tested here.

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“…Clearly, more research is needed to establish the best meta-omics and bioinformatic techniques as tools for identifying host-specific taxa and their genes for MST applications (23). Such studies will also establish whether or not metagenomic methods should be combined with conventional MST methods to obtain more accurate measures of fecal pollution in watersheds since qPCR provides absolute (vs. relative for typical metagenomics studies) abundances and generally has a lower limit of detection than metagenome shotgun sequencing (24, 25).…”

Section: Introductionmentioning

confidence: 99%

Metagenome-based comparisons of decay rates and host-specificity of fecal microbial communities for improved microbial source tracking

Suttner

Kim

et al. 2021

Preprint

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Fecal material in the environment is a primary source of pathogens that cause waterborne diseases and affect over a billion people worldwide. Microbial source tracking (MST) assays based on single genes (e.g., 16S rRNA) do not always provide the resolution needed to attribute fecal contamination sources. In this work, we used dialysis bag mesocosms simulating a freshwater habitat that were spiked separately with cow, pig, or human feces to monitor the decay of host-specific fecal signals over time with metagenomics, traditional qPCR, and culture-based methods. Sequencing of the host fecal communities used as inocula recovered 79 non-redundant metagenome-assembled genomes (MAGs) whose abundance patterns showed that the majority of the fecal community signal was not detectable in the mesocosm metagenomes after four days. Several MAGs showed high host specificity, and thus are promising candidates for biomarkers for their respective host type. Traditional qPCR methods varied in their correlation with MAG decay kinetics. Notably, the human-specific Bacteroides assay, HF183/BFDRev, consistently under-estimated fecal pollution due to not being present in all hosts and/or primer mismatches. This work provides new insights on the persistence and decay kinetics of host-specific gut microbes in the environment and identifies several MAGs as putative biomarkers for improved MST.

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Metagenomics as a Tool To Monitor Reclaimed-Water Quality

Cited by 17 publications

References 73 publications

Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types

Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types

Comparison of ultrafiltration and iron chloride flocculation in the preparation of aquatic viromes from contrasting sample types

Metagenome-based comparisons of decay rates and host-specificity of fecal microbial communities for improved microbial source tracking

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