The impact of sampling, PCR, and sequencing replication on discerning changes in drinking water bacterial community over diurnal time-scales

Santos, Quyen Melina Bautista-de los; Schroeder, Joanna L.; Blakemore, Oliver; Moses, Jonathan; Haffey, Mark; Sloan, William T.; Pinto, Ameet J.

doi:10.1016/j.watres.2015.12.010

Cited by 43 publications

(48 citation statements)

References 42 publications

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“…Thus, a relative abundance of a strain at 1% is considered to be a low abundance in a mixed population. Also, Methylobacterium species have been found to be present in drinking water in the United Kingdom [46,47] but they have not been found to be abundant in Glasgow tap water [40]. Here, only the case in which Methylobacterium was inoculated at the lowest relative abundance of 1% is reported.…”

Section: Inoculation Of Methylobacterium Into Drinking Watermentioning

confidence: 82%

“…The treatment steps that are followed in this treatment plant are: coagulation, rapid gravity filtration, chlorine disinfection, and orthophosphate dosing. The tap was flushed prior to sampling to avoid the effects of water stagnation in the premise plumbing at the sampling point [40]. The concentration of total chlorine of the drinking water sampled from the tap was measured immediately after its sampling using the USEPA DPD Method 8167 and a colorimeter (DR 900 Hach, Loveland, CO, USA) and was found to be 0.36 mg/L [41].…”

Section: Drinking Water Culturementioning

confidence: 99%

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A Keystone Methylobacterium Strain in Biofilm Formation in Drinking Water

Tsagkari

Keating

Couto

et al. 2017

Water

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Abstract:The structure of biofilms in drinking water systems is influenced by the interplay between biological and physical processes. Bacterial aggregates in bulk fluid are important in seeding biofilm formation on surfaces. In simple pure and co-cultures, certain bacteria, including Methylobacterium, are implicated in the formation of aggregates. However, it is unclear whether they help to form aggregates in complex mixed bacterial communities. Furthermore, different flow regimes could affect the formation and destination of aggregates. In this study, real drinking water mixed microbial communities were inoculated with the Methylobacterium strain DSM 18358. The propensity of Methylobacterium to promote aggregation was monitored under both stagnant and flow conditions. Under stagnant conditions, Methylobacterium enhanced bacterial aggregation even when it was inoculated in drinking water at 1% relative abundance. Laminar and turbulent flows were developed in a rotating annular reactor. Methylobacterium was found to promote a higher degree of aggregation in turbulent than laminar flow. Finally, fluorescence in situ hybridisation images revealed that Methylobacterium aggregates had distinct spatial structures under the different flow conditions. Overall, Methylobacterium was found to be a key strain in the formation of aggregates in bulk water and subsequently in the formation of biofilms on surfaces.

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Section: Inoculation Of Methylobacterium Into Drinking Watermentioning

confidence: 82%

Section: Drinking Water Culturementioning

confidence: 99%

A Keystone Methylobacterium Strain in Biofilm Formation in Drinking Water

Tsagkari

Keating

Couto

et al. 2017

Water

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“…Additionally, De bautista‐ los Santos et al. () using high throughput sequencing of 16S rRNA amplicons for microbial characterization of drink water observed that bias and variability inherent to the PCR amplification and sequencing steps is significant enough to mask differences between bacterial communities from replicate samples. In addition, the average size of the reads obtained in this study (454 bp for 16S amplicon sequencing and 233 bp for WGSM sequencing) and the hypervariable regions analyzed differed between both strategies, potentially leading to different accuracy levels in the phylogenetic affiliation and distinct microbial community profiles (Wang et al., ).…”

Section: Discussionmentioning

confidence: 99%

Microbial diversity of a full‐scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing

et al. 2017

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The 16S rRNA gene amplicon and whole‐genome shotgun metagenomic (WGSM) sequencing approaches were used to investigate wide‐spectrum profiles of microbial composition and metabolic diversity from a full‐scale UASB reactor applied to poultry slaughterhouse wastewater treatment. The data were generated by using MiSeq 2 × 250 bp and HiSeq 2 × 150 bp Illumina sequencing platforms for 16S amplicon and WGSM sequencing, respectively. Each approach revealed a distinct microbial community profile, with Pseudomonas and Psychrobacter as predominant genus for the WGSM dataset and Clostridium and Methanosaeta for the 16S rRNA gene amplicon dataset. The virome characterization revealed the presence of two viral families with Bacteria and Archaea as host, Myoviridae, and Siphoviridae. A wide functional diversity was found with predominance of genes involved in the metabolism of acetone, butanol, and ethanol synthesis; and one‐carbon metabolism (e.g., methanogenesis). Genes related to the acetotrophic methanogenesis pathways were more abundant than methylotrophic and hydrogenotrophic, corroborating the taxonomic results that showed the prevalence of the acetotrophic genus Methanosaeta. Moreover, the dataset indicated a variety of metabolic genes involved in sulfur, nitrogen, iron, and phosphorus cycles, with many genera able to act in all cycles. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) revealed that microbial community contained 43 different types of antibiotic resistance genes, some of them were associated with growth chicken promotion (e.g., bacitracin, tetracycline, and polymyxin).

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“…This approach has exploded in popularity with advances in sequencing throughput such that it is now possible to characterize numerous samples with thousands of sequences per sample. Many factors can impact how a natural community is represented by the sequencing data including the method of acquiring samples (4–8), storage conditions (4–6, 9–12), extraction methods (13), amplification conditions (8, 14, 15), sequencing method (15–17), and analytical pipeline (15, 18–20). The increased sampling depth that is now available relative to previous Sanger sequencing-based methods is expected to compound the impacts of an investigator’s choices and the interpretation of their results.…”

Section: Introductionmentioning

confidence: 99%

The impact of DNA polymerase and number of rounds of amplification in PCR on 16S rRNA gene sequence data

Sze

Schloss

2019

Preprint

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1 PCR amplification of 16S rRNA genes is a critical, yet under appreciated step in the generation 2 of sequence data to describe the taxonomic composition of microbial communities. Numerous 3 factors in the design of PCR can impact the sequencing error rate, the abundance of chimeric 4 sequences, and the degree to which the fragments in the product represent their abundance in 5 the original sample (i.e. bias). We compared the performance of high fidelity polymerases and 6 varying number of rounds of amplification when amplifying a mock community and human stool 7 samples. Although it was impossible to derive specific recommendations, we did observe general 8 trends. Namely, using a polymerase with the highest possible fidelity and minimizing the number 9 of rounds of PCR reduced the sequencing error rate, fraction of chimeric sequences, and bias. 10 Evidence of bias at the sequence level was subtle and could not be ascribed to the fragments' 11 fraction of bases that were guanines or cytosines. When analyzing mock community data, the 12 amount that the community deviated from the expected composition increased with rounds of PCR. 13 This bias was inconsistent for human stool samples. Overall the results underscore the difficulty 14 of comparing sequence data that are generated by different PCR protocols. However, the results 15 indicate that the variation in human stool samples is generally larger than that introduced by the 16 choice of polymerase or number of rounds of PCR. 17 Importance 18 A steep decline in sequencing costs drove an explosion in studies characterizing microbial 19 communities from diverse environments. Although a significant amount of effort has gone into 20 understanding the error profiles of DNA sequencers, little has been done to understand the 21 downstream effects of the PCR amplification protocol. We quantified the effects of the choice of 22 polymerase and number of PCR cycles on the quality of downstream data. We found that these 23 choices can have a profound impact on the way that a microbial community is represented in the 24 sequence data. The effects are relatively small compared to the variation in human stool samples, 25 however, care should be taken to use polymerases with the highest possible fidelity and to minimize 26 2 the number of rounds of PCR. These results also underscore that it is not possible to directly 27 compare sequence data generated under different PCR conditions. 28 3

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The impact of sampling, PCR, and sequencing replication on discerning changes in drinking water bacterial community over diurnal time-scales

Cited by 43 publications

References 42 publications

A Keystone Methylobacterium Strain in Biofilm Formation in Drinking Water

A Keystone Methylobacterium Strain in Biofilm Formation in Drinking Water

Microbial diversity of a full‐scale UASB reactor applied to poultry slaughterhouse wastewater treatment: integration of 16S rRNA gene amplicon and shotgun metagenomic sequencing

The impact of DNA polymerase and number of rounds of amplification in PCR on 16S rRNA gene sequence data

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