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Characterizing the Zebrafish Oral Microbiota Using 16S Sequencing

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Characterizing the Zebrafish Oral Microbiota Using 16S Sequencing Lauri Paulamäki CHARACTERIZING THE ZEBRAFISH ORAL MICROBIOTA USING 16S SEQUENCING Faculty of Medicine and Health Technology Master’s thesis May, 2019 PRO GRADU -TUTKIELMA Paikka: Tampereen yliopisto Lääketieteen ja terveysteknologian tiedekunta Tekijä: PAULAMÄKI, LAURI ILMARI Otsikko: Seeprakalan suun mikrobiston karakterisointi käyttäen 16S sekvensointia Sivumäärä: 58 sivua, 6 hakemistosivua Ohjaajat: Dosentti Mataleena Parikka ja Leena-Maija Vanha-aho, FT Tarkastajat: Apulaisprofessori Vesa Hytönen ja Dosentti Mataleena Parikka Aika: Toukokuu 2019 TIIVISTELMÄ Tässä pro gradu -tutkielmassani esittelen menetelmät, joita käytettiin ensimmäisissä yrityksissä määrittää taksonomisesti seeprakalan suun ja suoliston mikrobiston kolmesta seeprakalayksilöstä. Tavoitteena tutkimuksessa on auttaa selvittämään mahdollisuutta käyttää seeprakalaa malliorganismina suun mikrobiston ja sen tasapainon järkkymisen tutkimisessa ja hoitostrategioiden kehittämisessä. Tutkimuksessa käytettiin hyväksi mikrobistojen tutkimuksessa oletusarvoiseksi menetelmäksi vakiintunutta bakteerien ja arkkien ribosomaalisen pienen alayksikön RNA:ta koodaavan 16S geenin sekvensointia Illumina MiSeq sekvensaattorilla. Sekvensointikirjaston luomiseen kokeiltiin eri lähestymistapoja, joista lopulta päädyttiin käyttämään Illuminan valmistamaa kirjastonvalmistuspakkausta. Tulosten perusteella seeprakalan suun ja suolen mikrobistot vaikuttavat olevan samankaltaisia, mutta analyyseissa kuitenkin selkeästi omiksi kokonaisuuksiksi ryhmittyviä. Suun mikrobistossa runsaslukuisimpana esiintyivät firmikuutteihin kuuluvat peptostreptococcaceae heimon bakteerit ja toisiksi yleisimpinä proteobakteereihin lukeutuvat aeromonadaceae heimon edustajat. Suolen mikrobistossa kahden yleisimmän heimon järjestys on päinvastainen. Suun mikrobistot olivat tutkimuksen perusteella suolen mikrobistoja lajirikkaampia ja niiden määräsuhteet olivat myös tasaisemmin jakautuneet Shannonin indeksillä mitattuna. Tulosten perusteella seeprakalan käyttömahdollisuudet suun mikrobiston mallintamiseen vaikuttavat lupaavilta. ii MASTER’S THESIS Place: TAMPERE UNIVERSITY Faculty of Medicine and Health Technology Author: PAULAMÄKI, LAURI ILMARI Title: Characterising the zebrafish oral microbiota using 16S sequencing Pages: 58 pages, 6 index pages Supervisors: Docent Mataleena Parikka and Dr. Leena-Maija Vanha-aho Reviewers: Assistant professor Vesa Hytönen and Docent Mataleena Parikka Date: May 2019 ABSTRACT In this master’s thesis I present the methods that was used in the first efforts to taxonomically characterise the zebrafish oral and gut microbiotas from three individual zebrafish. Purpose of the study is to assess the use of the zebrafish as a model organism for studying the oral microbiota and for testing treatment strategies for microbiome disruption in the oral environment. In the study, sequencing of bacterial and archaeal ribosomal small subunit 16S RNA coding gene with Illumina MiSeq sequencer was used, a method that has been established as a standard tool in microbiome studies. Different approaches for creating the sequencing library were tested, of which the Illumina library preparation kit was eventually used for creating the sequencing library. Based on the results, the microbiotas in zebrafish oral and gut environments are rather similar, yet distinguishable. The most abundant microbes in the oral environment were bacteria from the Firmicutes phylum, which belongs to the Peptostreptococcaceae family and the second most abundant family was Aeromonadaceae that is part of the Proteobacteria phylum. In the gut microbiota two most abundant families were the same as in the oral environment, but in the opposite order. In oral microbiota there were higher taxonomic diversity than in the gut microbiota and the relative abundances between taxons were more evenly distributed when measured with Shannon’s index. Based on these results, the potential of the zebrafish for modelling oral microbiota seem promising. iii ACKNOWLEDGEMENTS I conducted this project as part of Infection Biology group in Faculty of Medicine and Health Technology of Tampere University. I am grateful for Mataleena Parikka for the opportunity to work with this wonderful group of people, and all the colleagues for discussing with me about the countless problems that I encountered and cheering me up when things felt overwhelming. Thanks for Leena-Maija for proofreading the literature review; I want to note that all the remaining miss- spellings and oddities are my mistakes. I would also like to thank Henna and Hanna for giving me advice for the experiments, Aleksandra for the help in the lab and the contagious positivity, Sonja for drawing the spectacular image of the biofilm and all the friends and family for helping me to have a break of the work when I needed it the most. iv TABLE OF CONTENTS 1 Abbreviations ............................................................................................................................... 2 2 Introduction ................................................................................................................................. 3 3 Literature review .......................................................................................................................... 5 3.1 Microbiomes .......................................................................................................................... 5 3.1.1 The oral microbiome ...................................................................................................... 6 3.2 The effect of the microbiome on health ............................................................................... 8 3.2.1 Overview ........................................................................................................................ 8 3.2.2 Dysbiosis ......................................................................................................................... 8 3.2.3 Oral dysbiosis ................................................................................................................. 9 3.2.4 Possibilities in medicine ............................................................................................... 11 3.2.5 Animal models of dysbiosis .......................................................................................... 12 3.3 Choice of methods .............................................................................................................. 13 3.3.1 Zebrafish as a model organism .................................................................................... 14 3.3.2 The zebrafish in microbiome studies ........................................................................... 15 3.3.3 16S sequencing ............................................................................................................ 16 3.3.4 lllumina MiSeq ............................................................................................................. 16 3.3.5 Data analysis; OTUs versus ASVs ................................................................................. 17 4 Aims of the study ....................................................................................................................... 19 5 Materials and methods .............................................................................................................. 20 5.1 Sample Collection ................................................................................................................ 20 5.2 DNA extraction methods ..................................................................................................... 20 5.2.1 Phenol-chloroform extraction ..................................................................................... 20 5.2.2 Comparison of DNA extraction methods ..................................................................... 21 5.3 Primer design....................................................................................................................... 21 5.3.1 Testing of primer pairs ................................................................................................. 22 v 5.4 Library creation in single PCR run ....................................................................................... 23 5.4.1 Mycobacterium marinum culture for positive control ................................................ 23 5.4.2 Annealing temperature optimization .......................................................................... 23 5.5 Library creation in single PCR run with two annealing temperatures ................................ 23 5.5.1 Annealing gradient experiment ................................................................................... 24 5.5.2 Additive PCR panel ....................................................................................................... 24 5.6 Exonuclease treatment ....................................................................................................... 25 5.7 Nextera XT Library Preparation Kit ...................................................................................... 26 5.7.1 AMPure XP Bead purification ....................................................................................... 26 5.7.2 Fragment analyzer ....................................................................................................... 26 5.8 Sequencing .......................................................................................................................... 27 5.8.1
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