Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

Moen, Aina Elisabeth Fossum; Tannæs, Tone M.; Vatn, Simen; Ricanek, Petr; Vatn, Morten H.; Jahnsen, Jørgen

doi:10.1186/s13104-016-2110-7

Cited by 27 publications

(27 citation statements)

References 26 publications

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“…Samples were then transported from Aberdeen to Dundee for processing. Upon arrival the sample were thawed and suspended in phosphate buffer saline where genomic DNA was extracted and purified using the DNA/RNA All Prep kit (Qiagen, Hilden, Germany) and stored according to manufacturer's instructions. Standard protocol, 16S Metagenomic Sequencing Library Preparation Guide (Illumina), was followed to prepare sequencing libraries targeting the variable V3 and V4 regions of the 16S rRNA gene and paired‐end sequencing was performed on the MiSeq System (Illumina).…”

Section: Methodsmentioning

confidence: 99%

Microbiome characteristics of induced sputum compared to bronchial fluid and upper airway samples

Warris

Turner

2018

Pediatric Pulmonology

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Objective: The study of the community of microorganisms (the microbiota) in the lower airways in children is restricted to opportunistic sampling in children undergoing elective general anaesthetic. Here we tested the hypothesis that induced sputum is a valid alternative to directly sampling the lower airways to study lower airway microbiota. Methods: Children scheduled for elective operations were recruited. Pre-operatively a sample of induced sputum was obtained. After anaesthesia was induced, a bronchial brushing and swabs of the upper respiratory tract were obtained. Bacterial community analysis was performed by amplification of the V3-V4 16S rRNA gene region. Results: Twenty children were recruited, mean age 10.7 years. Induced sputum samples were obtained from 12 children, bronchial brushing from 14 and nasal, mouth, and throat samples in 15, 16, and 17 children. The profile of bacterial communities was similar in the mouth, throat, and sputum samples with the nose and bronchial samples being different. Actinobacteria species dominated the nose and mouth, Fusobacteria were the dominant species in the throat and sputum while Proteobacteria species dominated in bronchial samples. Forty-one percent of detected bacteria in bronchial samples were unclassified. Bacterial communities from the mouth, throat, and induced sputum were tightly clustered and were distinct from nose and those found in bronchial communities. Conclusions: Induced sputum may not be a valid surrogate for microbiome assessment of the lower airways in all individuals. Many bacteria in bronchial samples were not recognized by standard testing, suggesting that our understanding of the lower airway microbiota in children remains rudimentary. K E Y W O R D S bronchial fluid, child, microbiota, sputum

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Section: Methodsmentioning

confidence: 99%

Microbiome characteristics of induced sputum compared to bronchial fluid and upper airway samples

Warris

Turner

2018

Pediatric Pulmonology

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“…Addition of mechanical lysis has improved isolation of Gram-positive bacteria (4,9,16), without impairing the isolation of Gram-negative bacteria (19). Additionally, enzymatic lysis with mutanolysin may help to identify more Gram-positive bacteria (4,20). The ultimate goal is to increase the bacterial to human DNA ratio and have a DNA isolate that closely reflects the bacterial composition of the sample.…”

Section: Another Major Advantage Of This Technique Is the Ability To mentioning

confidence: 99%

“…Importantly, fine-tuning of bead-beating speed and duration may be required for each specific bead-beater. It has been questioned whether bead-beating improves bacterial DNA isolation from tissues (36), because it may contribute to some level of DNA degradation (20,36). However, according to more recent studies, bead-beating does not cause DNA shearing (6,10) and results in identification of extra species in tissue isolates (18).…”

Section: The Inclusion Of Bead-beating Enhanced Isolation Of All Bactmentioning

confidence: 99%

Optimized DNA isolation method for microbiome analysis of human tissues

Bruggeling

Garza

Achouiti

et al. 2020

Preprint

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Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, developments in the field of tissue microbiomes have been hampered by the presence of host DNA in isolates which interferes with the analysis of the bacterial content. Here, we present a DNA isolation protocol from tissue samples including reduction of host DNA without distortion of microbial abundance profiles. We evaluated which concentrations of Triton and saponin lyse host cells and leave bacterial cells intact, which was combined with DNAse treatment to deplete released host DNA. We applied our protocol to extract microbial DNA from ex vivo and in vivo acquired human colon biopsies (∼2-5 mm in size) and assessed the relative abundance of bacterial and human DNA by qPCR. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ-Proteobacteria and Actinobacteria. Our protocol combined with shotgun metagenomic sequencing revealed a colon tissue microbiome profile with a Shannon diversity index of 3.2 and an UniFrac distance of 0.54, which is comparable to reported numbers based on amplicon sequencing. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of rigid Gram-positive bacteria without depleting the more sensitive Gram-negative bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

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“…Samples were then transported from Aberdeen to Dundee for processing. Upon arrival the sample had thawed and were suspended in phosphate buffer saline where genomic DNA was extracted and purified using the DNA/RNA All Prep kit 14 (Illumina), was followed to prepare sequencing libraries targeting the variable V3 and V4 regions of the 16S rRNA gene and paired-end sequencing was performed on the MiSeq System (Illumina). We followed sampling and controls procedures described by the Earlham Institute (http://www.earlham.ac.uk/sites/default/files/docs/Genomic%20Services/Sample%20Guide lines%20Aug17.pdf) and Illumina (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/1 6s-metagenomic-library-prep-guide-15044223-b.pdf which are designed to minimize the risk for contamination.…”

Section: Dna Extraction and Sequencingmentioning

confidence: 99%

“…Samples were then transported from Aberdeen to Dundee for processing. Upon arrival the sample had thawed and were suspended in phosphate buffer saline where genomic DNA was extracted and purified using the DNA/RNA All Prep kit 14 (minimum combined abundance was set at 10). Summary of processed sequence data is described in Table S1.…”

Section: Dna Extraction and Sequencingmentioning

confidence: 99%

Microbiome Characteristics of Induced Sputum compared to Bronchial Fluid and Upper Airway Samples

Turner¹,

Warris²,

An³

2018

Paediatric Respiratory Infection and Immun.

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Objective. The study of the community of microorganisms (the microbiota) in the lower airways in children is restricted to opportunistic sampling in children undergoing elective general anaesthetic. Here we tested the hypothesis that induced sputum is a valid alternative to directly sampling the lower airways to study lower airway microbiota. Methods. Children scheduled for elective operations were recruited. Pre-operatively a sample of induced sputum was obtained. After anaesthesia was induced, a bronchial brushing and swabs of the upper respiratory tract were obtained. Bacterial community analysis was performed by amplification of the V3-V4 16S rRNA gene region. Results. Twenty children were recruited, mean age 10.7 years. Induced sputum samples were obtained from 12 children, bronchial brushing from 14 and nasal, mouth and throat samples in 15, 16 and 17 children. The profile of bacterial communities was similar in the mouth, throat and sputum samples with the nose and bronchial samples being different. Actinobacteria species dominated the nose and mouth, Fusobacteria were the dominant species in the throat and sputum whilst Proteobacteria species dominated in bronchial samples. Forty-one percent of detected bacteria in bronchial samples were unclassified. Bacterial communities from the mouth, throat and induced sputum were tightly clustered and were distinct from nose and those found in bronchial communities. Conclusions. Induced sputum may not be a valid surrogate for microbiome assessment of the lower airways in all individuals. Many bacteria in bronchial samples were not recognised by standard testing, suggesting that our understanding of the lower airway microbiota in children remains rudimentary.

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Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

Cited by 27 publications

References 26 publications

Microbiome characteristics of induced sputum compared to bronchial fluid and upper airway samples

Microbiome characteristics of induced sputum compared to bronchial fluid and upper airway samples

Optimized DNA isolation method for microbiome analysis of human tissues

Microbiome Characteristics of Induced Sputum compared to Bronchial Fluid and Upper Airway Samples

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