University of the Pacific Scholarly Commons University of the Pacific Theses and Dissertations Graduate School 2019 Transcriptome analysis for novel peptide in breeding gland of Hymenochirus boettgeri Minjin Ko University of the Pacific, [email protected] Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds Part of the Biology Commons Recommended Citation Ko, Minjin. (2019). Transcriptome analysis for novel peptide in breeding gland of Hymenochirus boettgeri. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/3615 This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. 1 TRANSCRIPTOME ANALYSIS FOR NOVEL PEPTIDES IN BREEDING GLAND OF HYMENOCHIRUS BOETTGERI By Minjin Ko A Thesis Submitted to the Graduate School In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE College of the Pacific Biological Sciences University of the Pacific Stockton, California 2019 2 TRANSCRIPTOME ANALYSIS FOR NOVEL PEPTIDES IN BREEDING GLAND OF HYMENOCHIRUS BOETTGERI By Minjin Ko APPROVED BY: Dissertation Advisor: Eric Thomas, Ph.D. Committee Member: Jane Khudyakov, Ph.D. Committee Member: Doug Risser, Ph.D. Committee Member: Ajna Rivera, Ph.D. Department Co-Chairs: Lisa Wrischnik, Ph.D. and Eric Thomas, Ph.D. Dean of the Graduate School: Thomas Naehr, Ph.D. 3 DEDICATION I dedicate this to my parents. 4 Transcriptome Analysis for Novel Peptides in Breeding Gland of Hymenochirus Boettgeri Abstract By Minjin Ko University of the Pacific 2019 The primary goal of the project was to find nucleotide sequences potentially encoding a pheromone from the breeding gland of Hymenochirus boettgeri . The reasons in searching for the sequence of a pheromone were to better understand the organism and to use the information for application in reproduction of other species. Due to climate change and rampant deforestation, such as in Africa’s Congo Basin, many amphibian species are being threatened. With these increasing threats, a viable option for the future may be breeding in captivity for the amphibian species. Pheromone characterization from the breeding gland of Hymenochirus boettgeri may help with better understanding of reproduction in other species. Overall, five candidate signaling peptides that have the potential to be a pheromone from the breeding gland were found with transcriptome assembly and annotation of the breeding gland. The project focused on obtaining transcriptome data for the breeding gland by 1) RNA sequencing 2) de novo transcriptome assembly and 3) transcriptome annotation. The transcriptome of the breeding gland is now available for future proteomic studies. 5 TABLE OF CONTENTS LIST OF TABLES .............................................................................................................7 LIST OF FIGURES ............................................................................................................8 GLOSSARY .......................................................................................................................9 CHAPTER 1. Introduction ............................................................................................................10 2. Methods..................................................................................................................20 RNA Isolation ............................................................................................20 RNA Quality Assessment ..........................................................................21 Sequencing Library Preparation ................................................................22 High-Throughput Sequencing ....................................................................22 Sequencing Data Quality Assessment and Trimming ...............................22 De novo Transcriptome Assembly and Annotation ...................................23 Differential Gene Expression Analysis ......................................................23 Functional Enrichment Analysis ................................................................24 Analysis of Upregulated BG Transcripts ...................................................24 3. Results ....................................................................................................................25 RNA Isolation ............................................................................................25 Sequencing Data Quality Assessment .......................................................28 Transcriptome Assembly ...........................................................................30 Differential Gene Expression in BG relative to L .....................................33 Analysis of Unannotated Transcripts .........................................................36 6 4. Discussion ..............................................................................................................41 REFERENCES ..................................................................................................................50 7 LIST OF TABLES Table 1. Assembly statistics of the two Trinity de novo assemblies, BG and BG+L ..........31 2. Top ten enriched KEGG Pathways in the BG transcriptome, shown in descending order of number of transcripts mapping to each category ..................32 3. Top ten enriched GO Biological Processes in the BG transcriptome, shown in descending order of number of transcripts mapping to each category ..................32 4. Three specific genes that belong to a different category of the top ten overrepresented functional categories in the BG transcriptome .......................................................33 5. The top 15 genes upregulated in BG compared to L shown in descending order of log-transformed fold change values (log2 FC, BG/L) ...........................................34 6. KEGG categories overrepresented in set of genes upregulated in BG ...................35 7. GO BP categories overrepresented in set of genes upregulated in BG ..................36 8. Two unannotated transcripts with high BG transcript counts relative to L transcript counts .....................................................................................................37 9. The sequences of the two highlighted unannotated transcripts ..............................37 10. The max score, e-value, % identity, and predicted name of the two unannotated transcripts ...............................................................................................................38 11. The predicted amino acid sequence and the number of amino acids of the two unannotated transcripts ..........................................................................................38 12. Two annotated transcripts out of the top 30 unannotated differentially expressed genes using BLASTn .............................................................................................39 13. The five amino acid sequences with signal peptides of five transcripts that do not have any BLASTn hits ...................................................................................................40 8 LIST OF FIGURES Figure 1. Courtship behavior of male Cynops pyrrhogaster .................................................. 11 2. Male red-legged salamanders, Plethodon shermani , release courtship pheromones to persuade non-receptive females ......................................................12 3. Female Hymenochirus positive chemotaxis ............................................................14 4. H. boettgeri has different types of glands in skin ..................................................15 5. Illustration of breeding gland structure in H. boettgeri ................................................. 16 6. Bioanalyzer data of the first two RNA samples of breeding gland and skin ..........26 7. Bioanalyzer data of ladder and two samples of breeding gland (BG) and liver (L) ... .................................................................................................................................28 8. Per base sequence quality of the left read (R1) of breeding gland ..........................29 9. Mean per sequence quality score of R1 (left read) of breeding gland ....................30 9 GLOSSARY • De novo transcriptome assembly: the re-construction of a transcriptome from short sequenced cDNA reads without a reference genome. • High-throughput sequencing: the generation of hundreds of millions of DNA sequences in a short period of time. • NanoDrop: an instrument used to check RNA quality by using ultraviolet absorbance. The main wavelengths of absorbance are 260 nm, 280 nm, and 230 nm. 260 nm measures the amount of nucleic acids, 280 nm measures the amount of protein (aromatic amino acids) and 230 nm measures the presence of other contaminants. The ideal ratios of wavelengths are 1.8-2.2 for A260/A280 and greater than 1.7 for A260/A230. A factor to be wary of is that absorbance measurements can overestimate the quantity of interested nucleic acids. This is because all nucleic acids absorb at 260nm and instruments such as the Nanodrop quantify all the nucleic acids together without distinction. Some contaminants are also absorbed at 260 nm, which can also contribute to overestimation and mislead in the purity of nucleic acid by