Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing

Sui, Hui-Yu; Weil, Ana A.; Nuwagira, Edwin; Qadri, Firdausi; Ryan, Edward T.; Mezzari, Melissa P.; Phipatanakul, Wanda; Lai, Peggy S.

doi:10.3389/fmicb.2020.00953

Cited by 49 publications

(50 citation statements)

References 66 publications

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“…For the microbiome, taxa, genes, and pathways present in <5% of samples (i.e., fewer than four samples here) were excluded to filter out potentially spurious features due to sequencing or classification error ( 53 ). ICCs were calculated based on the square root of the relative abundances of the top 4 phyla (i.e., Bacteroidetes , Firmicutes , Proteobacteria , and Actinobacteria ) and 15 microbial diversity metrics (observed richness, Shannon index, and Simpson index for α-diversity and the top PCoA component [PC1] for Bray-Curtis distance and Jaccard distance for β-diversity), including 5 at the species level, 5 at the gene level, and 5 at the pathway level.…”

Section: Methodsmentioning

confidence: 99%

Comparison of Fecal Collection Methods on Variation in Gut Metagenomics and Untargeted Metabolomics

Guan

Liu

et al. 2021

mSphere

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The choice of fecal collection method is essential for studying gut microbe-human interactions in large-scale population-based research. In this study, we examined the effects of fecal collection methods and storage time at ambient temperature on variations in the gut microbiome community composition; microbial diversity metrics at the species, gene, and pathway levels; antibiotic resistance genes; and metabolome profiling.

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Section: Methodsmentioning

confidence: 99%

Comparison of Fecal Collection Methods on Variation in Gut Metagenomics and Untargeted Metabolomics

Guan

Liu

et al. 2021

mSphere

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“…Several challenges have hindered the adoption of sequencing technologies in routine clinical practice for microbial keratitis, including sample collection methods that are frequently ineffective (Kaye et al, 2003) compounded by the low abundance of pathogens (Ung et al, 2020), high contamination from background host DNA or laboratory reagents (Gu, Miller & Chiu, 2019), lack of standardisation in sequencing and bioinformatics processing methods . Sampling and DNA extraction methodology significantly impact upon the downstream sequencing data in samples with low biomass (Douglas et al, 2020;Sui et al, 2020). Microbial cells within the sample must be sufficiently lysed to liberate its DNA content, and this is particularly challenging for thick-walled microorganisms whereby mechanical disruption method such as bead-beating in conjunction with heat, chemical or enzymatic treatment is required in the DNA extraction protocol (De Boer et al, 2010;Ojo-Okunola et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis

Low

Fuentes-Utrilla

Hodson

et al. 2021

PeerJ

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Background Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis. Methods Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods. Results We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results. Conclusion We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.

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“…Numerous studies evaluating different extraction platforms in terms of their viral qPCR performance have found that the choice of extraction platform has a major impact on the reliability of the diagnostic results [ 24 , 25 , 26 , 27 , 28 , 29 ]. Furthermore, nucleic acid extraction methods can also impact bacteriome profiles [ 30 , 31 , 32 ] as well as the detection of particular viruses with mNGS [ 16 , 23 , 27 , 29 ].…”

Section: Introductionmentioning

confidence: 99%

“…Other potential issues related to extraction methods are sample cross-contamination (contamination from one sample to another) [ 24 , 33 , 34 ] and contamination by sequences present in the environment [ 32 , 34 ] or in the molecular biology reagents (referred to as the kitome) [ 35 , 36 , 37 ]. In viral mNGS studies, these two aspects constitute a major concern and must be precisely evaluated [ 38 , 39 , 40 ].…”

Section: Introductionmentioning

confidence: 99%

Comparison of Nucleic Acid Extraction Methods for a Viral Metagenomics Analysis of Respiratory Viruses

et al. 2020

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Viral metagenomics next-generation sequencing (mNGS) is increasingly being used to characterize the human virome. The impact of viral nucleic extraction on virome profiling has been poorly studied. Here, we aimed to compare the sensitivity and sample and reagent contamination of three extraction methods used for viral mNGS: two automated platforms (eMAG; MagNA Pure 24, MP24) and the manual QIAamp Viral RNA Mini Kit (QIAamp). Clinical respiratory samples (positive for Respiratory Syncytial Virus or Herpes Simplex Virus), one mock sample (including five viruses isolated from respiratory samples), and a no-template control (NTC) were extracted and processed through an mNGS workflow. QIAamp yielded a lower proportion of viral reads for both clinical and mock samples. The sample cross-contamination was higher when using MP24, with up to 36.09% of the viral reads mapping to mock viruses in the NTC (vs. 1.53% and 1.45% for eMAG and QIAamp, respectively). The highest number of viral reads mapping to bacteriophages in the NTC was found with QIAamp, suggesting reagent contamination. Our results highlight the importance of the extraction method choice for accurate virome characterization.

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Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing

Cited by 49 publications

References 66 publications

Comparison of Fecal Collection Methods on Variation in Gut Metagenomics and Untargeted Metabolomics

Comparison of Fecal Collection Methods on Variation in Gut Metagenomics and Untargeted Metabolomics

Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis

Comparison of Nucleic Acid Extraction Methods for a Viral Metagenomics Analysis of Respiratory Viruses

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