Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels

Manzari, Caterina; Oranger, Annarita; Fosso, Bruno; Piancone, Elisabetta; Pesole, Graziano; D’Erchia, Anna Maria

doi:10.1099/mgen.0.000417

Cited by 14 publications

(15 citation statements)

References 54 publications

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“…To verify the significance of the results, 24 specimens were prepared by dividing each sample into eight identical sampling tubes. We confirmed the integrity (e.g., DNA degradation, concentration, and purity) of the extracted microbial genomic DNA by considering the extraction of high-quality metagenomic DNA may affect its accurate microbial quantification 24 . In the case of the LoopSeq 16S Microbiome SSC 24-Plex kit, it consisted of a multiplex workflow that pools at least 24 samples into a single tube per sequencing run, thus, we performed the 16S V3–V4 amplicon sequencing procedure with the same samples.…”

Section: Resultssupporting

confidence: 55%

The effect of taxonomic classification by full-length 16S rRNA sequencing with a synthetic long-read technology

Jeong

Yun

Mun

et al. 2021

Sci Rep

102

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Characterizing the microbial communities inhabiting specimens is one of the primary objectives of microbiome studies. A short-read sequencing platform for reading partial regions of the 16S rRNA gene is most commonly used by reducing the cost burden of next-generation sequencing (NGS), but misclassification at the species level due to its length being too short to consider sequence similarity remains a challenge. Loop Genomics recently proposed a new 16S full-length-based synthetic long-read sequencing technology (sFL16S). We compared a 16S full-length-based synthetic long-read (sFL16S) and V3-V4 short-read (V3V4) methods using 24 human GUT microbiota samples. Our comparison analyses of sFL16S and V3V4 sequencing data showed that they were highly similar at all classification resolutions except the species level. At the species level, we confirmed that sFL16S showed better resolutions than V3V4 in analyses of alpha-diversity, relative abundance frequency and identification accuracy. Furthermore, we demonstrated that sFL16S could overcome the microbial misidentification caused by different sequence similarity in each 16S variable region through comparison the identification accuracy of Bifidobacterium, Bacteroides, and Alistipes strains classified from both methods. Therefore, this study suggests that the new sFL16S method is a suitable tool to overcome the weakness of the V3V4 method.

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Section: Resultssupporting

confidence: 55%

The effect of taxonomic classification by full-length 16S rRNA sequencing with a synthetic long-read technology

Jeong

Yun

Mun

et al. 2021

Sci Rep

102

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“…The presence of chlorine is known to negatively affect planktonic bacteria’s growth in DWDSs [ 18 , 51 ]. In terms of the quality of extracted DNA, gel electrophoresis ( S1 Fig ) shows the prominent absence of intact DNA, indicating low DNA integrity caused by degradation [ 52 , 53 ].…”

Section: Discussionmentioning

confidence: 99%

“…A rarefaction curve is also useful to assess whether the maximum sample diversity has been achieved and down amplification to lower reads (e.g., 10-100 reads/gene copies) is justifiable. However, low DNA concentration or any genetic markers have been demonstrated to give high false-negative results, increased false discovery and artefactual results [44,52] that is likely to be the case for sequencing-based studies in chlorinated drinking water [29]. We found that many reported drinking water microbiome studies from chlorinated systems (including 16s rRNA amplicon sequencing/ metabarcoding, metagenomics) [20,[56][57][58][59][60][61][62][63][64] often exclude raw experimental data of extracted DNA concentrations that is crucial to assess the quality of the sequencing results.…”

Section: No Impact Of Water Volume On Dna Recoverymentioning

confidence: 99%

Evaluation of DNA extraction yield from a chlorinated drinking water distribution system

et al. 2021

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Desalination technology based on Reverse Osmosis (RO) membrane filtration has been resorted to provide high-quality drinking water. RO produced drinking water is characterized by a low bacterial cell concentration. Monitoring microbial quality and ensuring membrane-treated water safety has taken advantage of the rapid development of DNA-based techniques. However, the DNA extraction process from RO-based drinking water samples needs to be evaluated regarding the biomass amount (filtration volume) and residual disinfectant such as chlorine, as it can affect the DNA yield. We assessed the DNA recovery applied in drinking water microbiome studies as a function of (i) different filtration volumes, (ii) presence and absence of residual chlorine, and (iii) the addition of a known Escherichia coli concentration into the (sterile and non-sterile, chlorinated and dechlorinated) tap water prior filtration, and directly onto the (0.2 μm pore size, 47 mm diameter) mixed ester cellulose membrane filters without and after tap water filtration. Our findings demonstrated that the co-occurrence of residual chlorine and low biomass/cell density water samples (RO-treated water with a total cell concentration ranging between 2.47 × 102–1.5 × 103 cells/mL) failed to provide sufficient DNA quantity (below the threshold concentration required for sequencing-based procedures) irrespective of filtration volumes used (4, 20, 40, 60 L) and even after performing dechlorination. After exposure to tap water containing residual chlorine (0.2 mg/L), we observed a significant reduction of E. coli cell concentration and the degradation of its DNA (DNA yield was below detection limit) at a lower disinfectant level compared to what was previously reported, indicating that free-living bacteria and their DNA present in the drinking water are subject to the same conditions. The membrane spiking experiment confirmed no significant impact from any potential inhibitors (e.g. organic/inorganic components) present in the drinking water matrix on DNA extraction yield. We found that very low DNA content is likely to be the norm in chlorinated drinking water that gives hindsight to its limitation in providing robust results for any downstream molecular analyses for microbiome surveys. We advise that measurement of DNA yield is a necessary first step in chlorinated drinking water distribution systems (DWDSs) before conducting any downstream omics analyses such as amplicon sequencing to avoid inaccurate interpretations of results based on very low DNA content. This study expands a substantial source of bias in using DNA-based methods for low biomass samples typical in chlorinated DWDSs. Suggestions are provided for DNA-based research in drinking water with residual disinfectant.

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“…Digital PCR was performed to determine the total number of 16S copies by using universal primers targeting the V5–V6 regions of 16S rDNA (primer sequences: forward, B-V5□:□5′-ATTAGATACCCYGGTAGTCC-3′; reverse, A-V6, 5′-ACGAGCTGACGACARCCATG-3′), according to established procedures (52).…”

Section: Methodsmentioning

confidence: 99%

“…Digital PCR was performed to determine the total number of 16S copies by using universal primers targeting the V5-V6 regions of 16S rDNA (primer sequences: forward, B-V5 : 5′-ATTAGATACCCYGGTAGTCC-3′; reverse, A-V6, 5′-ACGAGCTGACGACARCCATG-3′), according to established procedures (52). Absolute quantification was performed using QuantaSoft version 7.4.1 software (Bio-Rad, Hercules, CA, USA,) and the negative/positive thresholds were set manually, excluding samples with a number of droplets <10 000.…”

Section: Ddpcr Experimentsmentioning

confidence: 99%

Natural and after colon washing fecal samples: the two sides of the coin for investigating the human gut microbiome

Piancone

Fosso

Robertis

et al. 2021

Preprint

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To date there are several studies focusing on the importance of gut microbiome for human health, however the selection of a universal sampling matrix representative of the microbial biodiversity associated to the gastrointestinal (GI) tract, still represents a challenge. Here we present a study in which, through a deep metabarcoding analysis of the 16S rRNA gene, we compared two sampling matrices, feces (F) and colonic lavage liquid (LL), in order to evaluate their accuracy to represent the complexity of the human gut microbiome. A training set of 37 volunteers was attained and paired F and LL samples were collected from each subject. A preliminary absolute quantification of total 16S rDNA, performed by droplet digital PCR (ddPCR), confirmed that sequencing and taxonomic analysis were performed on same total bacterial abundance obtained from the two sampling methods. The taxonomic analysis of paired samples revealed that, although specific taxa were predominantly or exclusively observed in LL samples, as well as other taxa were detectable only or were predominant in stool, the microbiomes of the paired samples F and LL in the same subject hold overlapping taxonomic composition. Moreover, LL samples revealed a higher biodiversity than stool at all taxonomic ranks, as demonstrated by the Shannon Index and the Inverse Simpson's Index. We also found greater inter-individual variability than intra-individual variability in both sample matrices. Finally, functional differences were unveiled in the gut microbiome detected in the F and LL samples. A significant overrepresentation of 22 and 13 metabolic pathways, mainly occurring in Firmicutes and Proteobacteria, was observed in gut microbiota detected in feces and LL samples, respectively. This suggests that LL samples may allow for the detection of microbes adhering to the intestinal mucosal surface as members of the resident flora that are not easily detectable in stool, most likely representative of a diet-influenced transient microbiota. This first comparative study on feces and LL samples for the study of the human gut microbiome demonstrates that the use of both types of sample matrices may represent a possible choice to obtain a more complete view of the human gut microbiota in response to different biological and clinical questions.

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Accurate quantification of bacterial abundance in metagenomic DNAs accounting for variable DNA integrity levels

Cited by 14 publications

References 54 publications

The effect of taxonomic classification by full-length 16S rRNA sequencing with a synthetic long-read technology

The effect of taxonomic classification by full-length 16S rRNA sequencing with a synthetic long-read technology

Evaluation of DNA extraction yield from a chlorinated drinking water distribution system

Natural and after colon washing fecal samples: the two sides of the coin for investigating the human gut microbiome

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