Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

Walker, Sidney P.; Barrett, Maurice P. J.; Hogan, Glenn; Bueso, Yensi Flores; Claesson, Marcus J.; Tangney, Mark

doi:10.1038/s41598-020-73403-7

Cited by 36 publications

(30 citation statements)

References 24 publications

(19 reference statements)

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“…In contrast, bacterial concentrations can rise up to 6 log 10 cfu/mL, or even higher, in mastitis cases (Fernańdez et al, 2014) or if milk is collected through the use of non-sterile pumps (Boo et al, 2001;Brown et al, 2005;Marıń et al, 2009;Jimeńez et al, 2017). At present, cell count methods, from classic counting chambers to flow cytometry, are the best methods for an accurate quantification of (live) bacterial cells in milk while quantitative PCR methods usually lead to an overestimation due to the presence of dead bacterial cells, exosomes and/or free bacterial DNA but, also, to mispriming with human DNA (Walker et al, 2020), and copy number bias (Veťrovskýand Baldrian, 2013;Louca et al, 2018).…”

Section: The Composition Of the Human Milk Microbiotamentioning

confidence: 99%

The Microbiota of the Human Mammary Ecosystem

Fernández

Pannaraj

Rautava

et al. 2020

Front. Cell. Infect. Microbiol.

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Human milk contains a dynamic and complex site-specific microbiome, which is not assembled in an aleatory way, formed by organized microbial consortia and networks. Presence of some genera, such as Staphylococcus, Streptococcus, Corynebacterium, Cutibacterium (formerly known as Propionibacterium), Lactobacillus, Lactococcus and Bifidobacterium, has been detected by both culture-dependent and culture-independent approaches. DNA from some gut-associated strict anaerobes has also been repeatedly found and some studies have revealed the presence of cells and/or nucleic acids from viruses, archaea, fungi and protozoa in human milk. Colostrum and milk microbes are transmitted to the infant and, therefore, they are among the first colonizers of the human gut. Still, the significance of human milk microbes in infant gut colonization remains an open question. Clinical studies trying to elucidate the question are confounded by the profound impact of non-microbial human milk components to intestinal microecology. Modifications in the microbiota of human milk may have biological consequences for infant colonization, metabolism, immune and neuroendocrine development, and for mammary health. However, the factors driving differences in the composition of the human milk microbiome remain poorly known. In addition to colostrum and milk, breast tissue in lactating and non-lactating women may also contain a microbiota, with implications in the pathogenesis of breast cancer and in some of the adverse outcomes associated with breast implants. This and other open issues, such as the origin of the human milk microbiome, and the current limitations and future prospects are addressed in this review.

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Section: The Composition Of the Human Milk Microbiotamentioning

confidence: 99%

The Microbiota of the Human Mammary Ecosystem

Fernández

Pannaraj

Rautava

et al. 2020

Front. Cell. Infect. Microbiol.

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“…As the biopsies under study are low-biomass specimens, it was necessary to remove human-genome-aligning reads 14 , and ensure that any biological signal was not distorted by environmental contaminants or by inter-patient variation. SourceTracker (v1.0) 15 indicated low-to-moderate levels of contamination, which was subsequently removed with Decontam (v1.0.0) 16 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The bioinformatic contamination control tools Decontam (v1.0.0) 16 and SourceTracker (v1.0) 15 were used, according to published guidelines 9 , 14 , to retrospectively assess and remove contamination, based on sequencing data from negative controls.…”

Section: Methodsmentioning

confidence: 99%

Biopsy bacterial signature can predict patient tissue malignancy

Hogan

Eckenberger

Narayanen

et al. 2021

Sci Rep

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Considerable recent research has indicated the presence of bacteria in a variety of human tumours and matched normal tissue. Rather than focusing on further identification of bacteria within tumour samples, we reversed the hypothesis to query if establishing the bacterial profile of a tissue biopsy could reveal its histology / malignancy status. The aim of the present study was therefore to differentiate between malignant and non-malignant fresh breast biopsy specimens, collected specifically for this purpose, based on bacterial sequence data alone. Fresh tissue biopsies were obtained from breast cancer patients and subjected to 16S rRNA gene sequencing. Progressive microbiological and bioinformatic contamination control practices were imparted at all points of specimen handling and bioinformatic manipulation. Differences in breast tumour and matched normal tissues were probed using a variety of statistical and machine-learning-based strategies. Breast tumour and matched normal tissue microbiome profiles proved sufficiently different to indicate that a classification strategy using bacterial biomarkers could be effective. Leave-one-out cross-validation of the predictive model confirmed the ability to identify malignant breast tissue from its bacterial signature with 84.78% accuracy, with a corresponding area under the receiver operating characteristic curve of 0.888. This study provides proof-of-concept data, from fit-for-purpose study material, on the potential to use the bacterial signature of tissue biopsies to identify their malignancy status.

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“…Current studies may select primers that have low host genome amplification, such as the V4 region, but at the cost of reduced sensitivity in differentiating species such as L. acidophilus and L. crispatus (evidenced in Figure 2 ). There also exists a growing body of evidence highlighting the cross-amplification of host DNA using 16S rRNA primers ( Walker et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

Limitations of 16S rRNA Gene Sequencing to Characterize Lactobacillus Species in the Upper Genital Tract

O’Callaghan

Willner

Buttini

et al. 2021

Front. Cell Dev. Biol.

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The endometrial cavity is an upper genital tract site previously thought as sterile, however, advances in culture-independent, next-generation sequencing technology have revealed that this low-biomass site harbors a rich microbial community which includes multiple Lactobacillus species. These bacteria are considered to be the most abundant non-pathogenic genital tract commensals. Next-generation sequencing of the female lower genital tract has revealed significant variation amongst microbial community composition with respect to Lactobacillus sp. in samples collected from healthy women and women with urogenital conditions. The aim of this study was to evaluate our ability to characterize members of the genital tract microbial community to species-level taxonomy using variable regions of the 16S rRNA gene. Samples were interrogated for the presence of microbial DNA using next-generation sequencing technology that targets the V5–V8 regions of the 16S rRNA gene and compared to speciation using qPCR. We also performed re-analysis of published data using alternate variable regions of the 16S rRNA gene. In this analysis, we explore next-generation sequencing of clinical genital tract isolates as a method for high throughput identification to species-level of key Lactobacillus sp. Data revealed that characterization of genital tract taxa is hindered by a lack of a consensus protocol and 16S rRNA gene region target allowing comparison between studies.

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Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

Cited by 36 publications

References 24 publications

The Microbiota of the Human Mammary Ecosystem

The Microbiota of the Human Mammary Ecosystem

Biopsy bacterial signature can predict patient tissue malignancy

Limitations of 16S rRNA Gene Sequencing to Characterize Lactobacillus Species in the Upper Genital Tract

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