Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Anahtar, Melis N.; Bowman, Brittany; Kwon, Douglas S.

doi:10.3791/53939-v

Cited by 7 publications

(7 citation statements)

References 8 publications

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“…Except for the Xpert MTB/RIF Assay based tests, most TB molecular tests require extraction of nucleic acids in complex clinical specimen such as sputum [17,26]. The use of manual or automated extraction can create aerosols and present biosafety risk to laboratory staff, justifying the need for BSL3 laboratory equipped with Class II biological safety cabinets [25].…”

Section: Discussionmentioning

confidence: 99%

A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory

et al. 2023

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Section: Discussionmentioning

confidence: 99%

A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory

et al. 2023

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“…The procedure was repeated four times in order to collect the microbial samples, which were then subjected to DNA extraction using the FastDNA Soil Genomic DNA Extraction Kit (MP BIO, CA, USA). The V3V4 region of the 16S rRNA genes was amplified using forward primer 338F (ACTCCTACGGGAGGCAGCA) and reverse primer 806R (GGACTACHVGGGTWTCTAAT) . Amplicon sequencing was performed on the Illumina NovaSeq6000 platform with PE250 (Shanghai Personal Biotechnology Co., Ltd).…”

Section: Methodsmentioning

confidence: 99%

“…The V3V4 region of the 16S rRNA genes was amplified using forward primer 338F (ACTCCTACGGGAGGCAGCA) and reverse primer 806R (GGACTACHVGGGTWTCTAAT). 20 Amplicon sequencing was performed on the Illumina NovaSeq6000 platform with PE250 (Shanghai Personal Biotechnology Co., Ltd). Quality control, barcode removal, paired-end joining, and ASV generation were performed using the Dada2 pipeline.…”

Section: Chemicalsmentioning

confidence: 99%

Nano α-Fe₂O₃ Self-Assembled Hybrid Biofilm Boosts Hydrogen Autotrophic Denitrification in a Three-Dimensional Biofilm Electrode Reactor

et al. 2023

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The performance of three-dimensional biofilm electrode reactors (3D-BERs) in hydrogen autotrophic denitrification is determined by electroactive biofilms. Herein, a selfassembled hybrid biofilm (SAHB) is developed on granular activated carbon particle electrodes by incorporating nano α-Fe 2 O 3 . The influences of operating parameters such as current, influent concentration, and hydraulic retention time (HRT) on denitrification are investigated as well as the mechanisms of SAHB-improved denitrification. The results indicate that SAHB achieves a high autotrophic denitrification rate of 4.20 mg TN/L/h under 40 mA current, 100 mg/L nitrate, and 26 h HRT, outperforming the naturally formed biofilm by 23.53%. SAHB contains more microorganisms than naturally formed biofilms, revealing that nano α-Fe 2 O 3 is conducive to trapping free microorganisms on particle electrodes. SAHB enriches denitrification-related genes and increases corresponding enzyme activities (particularly nitrite reductase). According to metagenomic sequencing, nano α-Fe 2 O 3 addition at a current level of 40 mA appears to favor denitrification via the nirS-dependent pathway probably due to its high affinity for cytochrome C. Metagenomic binning reveals that the microorganism associated with Zoogloeaceae (Rhodocyclales order) in SAHB may play a pivotal role in hydrogen autotrophic denitrification. This work develops a novel denitrification system, furthering our understanding of hydrogen autotrophic denitrification in 3D-BERs.

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“…DNA was further purified with the DNeasy PowerClean Pro Cleanup Kit (QIAGEN) for 16S rRNA gene sequencing. Nucleic acids from the lavage pellets were extracted using a phenol-chloroform protocol [56], with RNAlater storage solution being removed by spinning down and washing with 1000 μL of PBS prior to bead beating. The 16S rRNA V4 gene region was amplified using the 515F/806R primer set and sequenced using forward reads on Illumina MiSeq as previously described [56].…”

Section: Microbial 16s Rrna Gene Sequencingmentioning

confidence: 99%

“…Nucleic acids from the lavage pellets were extracted using a phenol-chloroform protocol [56], with RNAlater storage solution being removed by spinning down and washing with 1000 μL of PBS prior to bead beating. The 16S rRNA V4 gene region was amplified using the 515F/806R primer set and sequenced using forward reads on Illumina MiSeq as previously described [56]. The 515F/806R primer set was used to ensure efficient amplification of Bifidobacteriaceae (including Gardnerella).…”

Section: Microbial 16s Rrna Gene Sequencingmentioning

confidence: 99%

Distinct cervical tissue-adherent and luminal microbiome communities correlate with mucosal host gene expression and protein levels in Kenyan sex workers

et al. 2023

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Background The majority of studies characterizing female genital tract microbiota have focused on luminal organisms, while the presence and impact of tissue-adherent ectocervical microbiota remain incompletely understood. Studies of luminal and tissue-associated bacteria in the gastrointestinal tract suggest that these communities may have distinct roles in health and disease. Here, we performed a multi-omics characterization of paired luminal and tissue samples collected from a cohort of Kenyan female sex workers. Results We identified a tissue-adherent bacterial microbiome, with a higher alpha diversity than the luminal microbiome, in which dominant genera overall included Gardnerella and Lactobacillus, followed by Prevotella, Atopobium, and Sneathia. About half of the L. iners-dominated luminal samples had a corresponding Gardnerella-dominated tissue microbiome. Broadly, the tissue-adherent microbiome was associated with fewer differentially expressed host genes than the luminal microbiome. Gene set enrichment analysis revealed that L. crispatus-dominated tissue-adherent communities were associated with protein translation and antimicrobial activity, whereas a highly diverse microbial community was associated with epithelial remodeling and pro-inflammatory pathways. Tissue-adherent communities dominated by L. iners and Gardnerella were associated with lower host transcriptional activity. Tissue-adherent microbiomes dominated by Lactobacillus and Gardnerella correlated with host protein profiles associated with epithelial barrier stability, although with a more pro-inflammatory profile for the Gardnerella-dominated microbiome group. Tissue samples with a highly diverse composition had a protein profile representing cell proliferation and pro-inflammatory activity. Conclusion We identified ectocervical tissue-adherent bacterial communities in all study participants of a female sex worker cohort. These communities were distinct from cervicovaginal luminal microbiota in a significant proportion of individuals. We further revealed that bacterial communities at both sites correlated with distinct host gene expression and protein levels. The tissue-adherent bacterial community could possibly act as a reservoir that seed the lumen with less optimal, non-Lactobacillus, bacteria.

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Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Cited by 7 publications

References 8 publications

A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory

A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory

Nano α-Fe₂O₃ Self-Assembled Hybrid Biofilm Boosts Hydrogen Autotrophic Denitrification in a Three-Dimensional Biofilm Electrode Reactor

Distinct cervical tissue-adherent and luminal microbiome communities correlate with mucosal host gene expression and protein levels in Kenyan sex workers

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Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Cited by 7 publications

References 8 publications

A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory

A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory

Nano α-Fe2O3 Self-Assembled Hybrid Biofilm Boosts Hydrogen Autotrophic Denitrification in a Three-Dimensional Biofilm Electrode Reactor

Distinct cervical tissue-adherent and luminal microbiome communities correlate with mucosal host gene expression and protein levels in Kenyan sex workers

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Nano α-Fe₂O₃ Self-Assembled Hybrid Biofilm Boosts Hydrogen Autotrophic Denitrification in a Three-Dimensional Biofilm Electrode Reactor