Fecal Bacterial Microbiota Study in a Mouse Model of Niemann Pick Type C1 Disease Antony Cougnoux, Section of Molecular Dysmorphology, NICHD

Microbiota or microbial flora or flora the microscopic living organisms of a particular region (soil, cloud, gut,...) include: bacteria (Escherichia, Mycobacterium, ) fungus (saccharomyces, candida....) archaebacteria or archaea protist (algae, plasmodium,...) virus (phage, enterovirus,...)

Microbiota analysis Studied in human since the late 50s Before the molecular biology era only cultivated bacteria ~25% of all class 16S PCR cloning and sanger sequencing of all the 16S gene cloned PCR-DGGE-fingerprinting 16S qPCR using specific primers Newest approach to microbiota analysis: High throughput sequencing

Why looking at the gut microbiota ? Not only quiescent microorganism here Wide spectrum of useful function Metabolism: vitamin production, polysaccharide processing, lipid absorption Immune function: maturate and educate the host immune system Hematopoietic function Neurotransmitter production Barrier for toxic compound ingested Full range of biological significance has not been elucidated Little is know about the function of the non bacterial organism (viral and fungal) of the gut microbiota, (immunomodulation for some fungi) Variation (disbiosis) in microbiota composition associated to several disease (Crohn`disease, diabetes, obesity, “autism”,…)

Normal gut microbiota and disbiosis Disbiosis: inbalance in the microbiota composition Shift in obesity

Fecal bacterial microbiota analysis Sample collection DNA extraction 16S amplification Purification Sequencing Depending on which part of the microbiota you are interested in the sample collection and DNA extraction is different Protocol dedicated to fungus, virus, archea

Mouse fecal sample collection Handle the mouse and directly collect in a 1.5mL vial (only if you have no space or no single use boxes and few mice) Wait 5-10 minutes Remove the mice Collect fecal pellet and store at -80 ⁰ C Or Do not re-use the same cup for a different mouse Throw after use if: paper cup Clean and autoclave if: pipet tip or other plastic box

DNA extraction Standard extraction time is 1 and a half hours to extract the DNA from a frozen pellet Follow the manufacturer instruction Qiagen is the most commonly used DNA extraction kit for fecal samples. Useful for comparison with other studies Depending on your population of interest several different kits could be used, DNA extracted from swabs require a different type of extraction (disrupting beads, DNA free reagent)

Other kit Mobio Ultra Clean ® Fecal DNA Isolation Kit (M) QIAamp ® DNA Stool Mini Kit (Q)(QA), FastDNA ® SPIN Kit (FSp), and FastDNA ® SPIN Kit for Soil (FSo). ZR Fecal DNA Isolation Kit™ (Zymo Research) QIAsymphony® Virus/Bacteria Mini Kit (Qiagen) QS

Kit comparison references Claassen et al., A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. Journal of Microbiological methods, August 2013, Pages 103–110 Kennedy et al., The Impact of Different DNA Extraction Kits and Laboratories upon the Assessment of Human Gut Microbiota Composition by 16S rRNA Gene Sequencing. PlosONE, February 24, Ariefdjohan et al., Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens. Nutrition Journal. 2010, 9:23. Henderson et al., Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities. Plos ONE September 11, DNA Extraction and Purification, Labome, Anandika Dhaliwal, updated in 2013

Microbiota analysis from other sites Much less DNA extracted from the sample External contamination Use only sterile reagent and consumable Clean the bench (Bleach or acetone and ethanol) Kit using beads are the most commonly used Online protocol for each type of sample from tree roots to clouds

16S PCR amplification 8F-1525r: historically used for 16S amplification and cloning 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) is the most commonly used forward primer for 16S sequencing 1525r (5’-AAGGAGGTGWTCCARCC-3’)is commonly used This PCR amplicon (1500 base pairs) will amplify the variable region of all phyla of bacteria used in taxonomic analysis PCR program 94 ⁰ C 5 minutes 94 ⁰ C 45 seconds 55 ⁰ C 45 seconds 72 ⁰ C 1 minute 30 seconds 20 cycles 72 ⁰ C 10 minutes 4 ⁰ C

PCR purification from large volume 0.8-1mL Single band at 1500bp MiniElute PCR purification Kit (QIAgen) BUT replace binding buffer by the DNA Gel extraction binding buffer OrangeG appear as a bright spot at the same size as primers Two or more band Isopropanol DNA precipitation (to reduce the volume to purify on gel) Gel extraction using MiniElute gel extraction kit (QIAgen) Purified product concentration and purity are analyzed using Nanodrop spectrophotometer (Thermo Scientific) Qubit Fluorometric quantitation (Invitrogen) 1% agarose gel separation PCR purification

Final Sample preparation Each sample should be store in an individual vial (Eppendorff 1.5mL) labeled Complete the MGL form for the microbiota sample Label use for the vial Description of the sample Sample concentration Picture of the purified 16S PCR product separated on 1% agarose gel as an additional file

Tube label Sample description Amplicon size Sample Volume Sample Concentration Purification Type Final Sample preparation