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DE NOVO TRANSCRIPTOME ASSEMBLY for the OLYMPIA OYSTER (OSTREA LURIDA), a SPECIES of CONSERVATIONAL CONCERN a University Thesis

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DE NOVO TRANSCRIPTOME ASSEMBLY for the OLYMPIA OYSTER (OSTREA LURIDA), a SPECIES of CONSERVATIONAL CONCERN a University Thesis DE NOVO TRANSCRIPTOME ASSEMBLY FOR THE OLYMPIA OYSTER (OSTREA LURIDA), A SPECIES OF CONSERVATIONAL CONCERN A University Thesis Presented to the Faculty of California State University, East Bay In Partial Fulfillment of the Requirements for the Degree Master of Science in Biological Science By Ashley Maynard June 2016 Copyright © 2016 by Ashley Maynard ii ABSTRACT The Olympia oyster, Ostrea lurida, is the only oyster native to the west coast of N. America. Once abundant, O. lurida is now considered functionally extinct and absence of Olympia oysters has contributed to a continual decline in the health of California’s valuable estuary ecosystems. Human-assisted reintroduction is one potential strategy to increase O. lurida population sizes and help restore Californian estuaries. Ideally, restoration would use genotypes capable of surviving future conditions. However, which O. lurida populations will be most tolerant of climate change, in particular low salinity, and therefore most suitable for use in reintroduction remains uncertain. RNA-Seq has emerged as a vital tool to understand ecological and evolutionary processes of non-model organisms. Using a novel RNA-Seq pipeline, a transcriptome for O. lurida was generated yielding 51,574 contigs and accounting for upwards of 10,000 unique genes. Quality control metrics including mean contig length (1664 bp), percent of reads mapped back to the reference transcriptome (78%), and the percent of annotated reads (49%), are similar to other non-model organism transcriptome assemblies, and offers a substantial improvement over existing sequence resources for O. lurida. In important future research, this transcriptome can be used to better understand how O. lurida populations manage environmental stress at the molecular level and help to identify which populations of O. lurida are ultimately best suited for reintroduction. iii DE NOVO TRANSCRIPTOME ASSEMBLY FOR THE OLYMPIA OYSTER (OSTREA LURIDA), A SPECIES OF CONSERVATIONAL CONCERN By Ashley Maynard Approved: Date: -/a- Lsr ' Tyler G. Evans , /Z~ • I s/[) /J~ Clistopher Baysdorfer iv ACKNOWLEDGEMENTS There are a lot of people that should be thanked for their support throughout this research and honestly I think making sure everyone is given their due is almost harder than writing all the research in the first place. For anyone who has completed or is even in the processing of scientific research, I am sure you can sympathize and relate to the vast amounts of tireless work that goes into it. I strongly believe that without the support of the following people I wouldn’t have ever been able to end this journey with my sanity intact. Firstly, I would like to express my sincere gratitude to my advisor Dr. Tyler Evans for his knowledge and guidance throughout my Master’s research. Besides my advisor, I would like to extend my thanks for the rest of my thesis committee: Dr. Christopher Baysdorfer and Dr. Brain Perry. I would also like to extend my thanks to my collaborators, Dr. Jill Bible and Dr. Eric Sanford, at UC Davis. If not for their amazing research and diligence this research would not have been possible. It should be mentioned that there are those that were not directly involved with this project but who have supplied me with confidence, strength and knowledge throughout my time at California State University East Bay and so, I would like to thank the staff and faculty of the Biology department. Additionally, I want to thank my friends from the BCP and Masters programs. It was the stimulating conversations, drinking nights and healthy competition that pushed me though all those bumps in the road so inherent to research. Lastly, but certainly not least, I want to thank my family. I love you all so very much and it really has been your support and love that have motivated me to press on even when I wanted to give up. Thank you all so very much, I am forever thankful. v TABLE OF CONTENTS Abstract .............................................................................................................................. iii Acknowledgements ............................................................................................................. v List of Figures .................................................................................................................. viii List of Tables ...................................................................................................................... x 1 Introduction ................................................................................................................. 1 1.1 Background .......................................................................................................... 1 1.2 Thesis Statement .................................................................................................. 8 1.3 Significance of Research ...................................................................................... 9 1.4 RNA Sequencing of Olympia Oysters ............................................................... 10 2 Experimental Procedures ........................................................................................... 14 2.1 Experimental Workflow ..................................................................................... 14 2.2 Collection and Salinity Challenge ...................................................................... 14 2.3 RNA Isolation .................................................................................................... 16 2.4 cDNA Library Construction ............................................................................... 18 2.5 Assessment of Library Quality ........................................................................... 26 2.6 RNA-Sequencing ............................................................................................... 27 2.7 Bioinformatics .................................................................................................... 29 2.8 Mapping Reads to De Novo Transcriptome ....................................................... 36 2.9 Phylogeny ........................................................................................................... 37 2.10 Comparison of Gene Ontology Among Bivalves .............................................. 38 3 Results ....................................................................................................................... 39 3.1 RNA Sequencing ................................................................................................ 39 3.2 Transcriptome Assembly.................................................................................... 39 3.3 Annotation .......................................................................................................... 45 4 Discussion .................................................................................................................. 49 4.1 De Novo Transcripome Assembly...................................................................... 49 4.2 Future Work ....................................................................................................... 65 References ......................................................................................................................... 68 Appendix A: RNA Isolation Information ......................................................................... 75 vi Appendix B: RNA-Seq Sample Lane Assignments.......................................................... 78 Appendix C: Assembly Parameters .................................................................................. 80 Appendix D: Raw Transcriptome Results ........................................................................ 83 Appendix E: Reads Mapped to Final Transcriptome ........................................................ 86 Appendix F: Genebank Sequences (16S) Used For Phylogeny Tree ............................... 87 vii LIST OF FIGURES Figure 1: O. lurida in natural environment in San Francisco Bay. ..................................... 2 Figure 2: The historic distribution of O. lurida .................................................................. 3 Figure 3: Climate processes, from global to estuarine outcomes ....................................... 6 Figure 4: Mortality results from salinity exposure among O. lurida populations .............. 8 Figure 5: Map of collection sites. ....................................................................................... 9 Figure 6: Maximum likelihood tree based on 16S subunit of rRNA. ............................... 12 Figure 7: Experimental workflow ..................................................................................... 14 Figure 8: O. lurida settled on experimental settlement plate. ........................................... 15 Figure 9: Traditional method of de novo assembly. ......................................................... 33 Figure 10: Stepwise method of de novo assembly. ........................................................... 35 Figure 11: Number of contigs comparison across four assembly levels .......................... 40 Figure 12: Mean contig length comparison across four assembly levels ......................... 41 Figure 13: N50 comparison across four assembly levels.................................................. 43 Figure 14: Nucleotide distribution among assembly levels. ............................................. 44 Figure 15: Percent of the sample reads mapped to the assembled transcriptome.
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