Details

Autor(en) / Beteiligte

• Klosinska, Karolina
• Reece, Emma
• Kenny, Elaine
• Renwick, Julie

Titel

Reducing human DNA bias in cystic fibrosis airway specimens for microbiome analysis

Ist Teil von

• Journal of microbiological methods, 2022-09, Vol.200, p.106540-106540, Article 106540

Ort / Verlag

Elsevier B.V

Erscheinungsjahr

2022

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• Next generation sequencing (NGS) has transformed our understanding of airway microbiology, however there are methodology limitations that require consideration. The presence of high concentrations of human DNA in clinical specimens can significantly impact sequencing of the microbiome, especially in low biomass samples. Here we compared three different methods (0.025% saponin, NEBNext Microbiome DNA enrichment kit, QIAamp DNA microbiome kit) for the reduction of human DNA from six CF sputum samples and determined the impact on the microbiome detected using 16S rRNA gene sequencing. Human DNA in undepleted CF sputum accounted for 94.3% of the total DNA. Saponin, the NEBNext kit and the QIAamp kit reduced human DNA levels by an average of 38.7%, 61.8% and 94.8%, respectively. None of the depletion methods reduced total bacterial DNA concentrations. QIAamp depletion did not influence taxa richness or alpha diversity however alterations to the core genera were noted following depletion. While all methods reduced human DNA in the CF sputum samples, the QIAamp DNA microbiome kit reduced Human DNA levels significantly while leaving bacterial DNA levels unchanged. Human DNA depletion in low biomass, human DNA-dense CF sputum samples is vital for improving bacterial resolution in the CF airway microbiome. •Human DNA in clinical samples can account for >90% of all DNA and can reduce resolution of the microbiome.•The CF airway microbiome is a low-density microbiome in a Human DNA rich environment and thus a complex sample to sequence.•The saponin method, the NEBNext microbiome DNA enrichment kit and the QIAamp DNA microbiome kit were compared.•The QIAamp DNA microbiome kit significantly depleted 94.8% ± 11.6% of human DNA while not impacting bacterial DNA yield.•Depletion did not impact diversity and Phylum level taxonomy however changes at the genus level were noted.