Comprehensive Gut, Oral, and Skin Microbiome Profiling

DNA extraction from the faecal samples is to be obtained by the use of the commercial kit, the PSP Spin Stool Kit (Stratec, USA), with an enzymatic and bead beating step to enhance DNA recovery and concentration. DNA extraction from the oral, vaginal and skin samples are to be obtained using the commercial kit, QIAamp DNA Mini Kit (Qiagen, USA) following previously published methodology.29 (link) DNA concentration is measured using the Qubit 2.0 Fluorometer (Life Technology, USA). Bacterial quantitative PCR analysis of samples will be undertaken to confirm the presence of bacterial DNA, prior to sequence analysis. PCR primers (926F30 and 1062R31 (link)), targeting total bacteria, will be performed using Quantstudio (Thermo Fisher Scientific) using SYBR Green chemistry (Roche). To rule out possible reagent and collection kit contamination, sample collection buffers and double distilled water will be included for DNA extraction, Qubit, qPCR and sequencing. Shotgun metagenomic libraries will be generated with the Illumina Nextera DNA Flex, sequenced on the NovaSeq 6000 Sequencing platform at the UNSW Ramaciotti Centre for Genomics.

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Susic D., Davis G., O' Sullivan A.J., McGovern E., Harris K., Roberts L.M., Craig M.E., Mangos G., Hold G.L., El-Omar E.M, & Henry A. (2020). Microbiome Understanding in Maternity Study (MUMS), an Australian prospective longitudinal cohort study of maternal and infant microbiota: study protocol. BMJ Open, 10(9), e040189.

Publication 2020

PSP Spin Stool Kit QIAamp DNA Mini Kit Qubit 2.0 Fluorometer Quantstudio SYBR Green chemistry Nextera DNA Flex NovaSeq 6000 Sequencing platform

Bacteria Bacterial dna Buffers Enzymatic Metagenomic Primers Sample collection Skin Stool Sybr green Vaginal

Corresponding Organization :

Other organizations : UNSW Sydney, Sutherland Hospital, Children's Hospital at Westmead, University of Sydney, The George Institute for Global Health

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Variable analysis

independent variables

Extraction method used (PSP Spin Stool Kit, QIAamp DNA Mini Kit)

dependent variables

DNA concentration
Presence of bacterial DNA

control variables

Sample collection buffers
Double distilled water

controls

Positive control: Inclusion of sample collection buffers and double distilled water for DNA extraction, Qubit, qPCR, and sequencing to rule out possible reagent and collection kit contamination.
Negative control: Not explicitly mentioned.

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