Presentation on theme: "Using transcriptomics to delineate the status of Millepora species in the Caribbean Sea Nikolaos V. Schizas, Dannise Ruiz Ramos, Ingrid Ortiz Gonzalez,"— Presentation transcript:
Using transcriptomics to delineate the status of Millepora species in the Caribbean Sea Nikolaos V. Schizas, Dannise Ruiz Ramos, Ingrid Ortiz Gonzalez, Audrey Majeske, Ernesto Weil University of Puerto Rico, Mayagüez Department of Marine Sciences
Background The hydrocoral genus Millepora consists of 19 species distributed in warm waters around the globe 12 are found in the Indo-Pacific, 7 in the Atlantic/Caribbean
Caribbean species of Millepora spp. Clockwise from the upper left: M. alcicornis, M. squarrosa, M. striata, and M. complanata
Millepora is an important reef framework builder in some reefs Millepora is a voracious plankton feeder, consuming up to 8 prey cm -2 day -1 Milleporids are superior space competitors over gorgonians Millepora are susceptible to bleaching Despite their abundance, geographical distribution and geological importance, the milleporids have seldom received attention in coral reef studies Background
(Photo:CCMA/NOAA) Millepora visually dominate some reefsMillepora overgrowing gorgonians
Photo credit- E. Weil 1) Are there reliable morphological characters that separate the species? 2) Do the different species/morphotypes of Caribbean Millepora represent genetically distinct taxa? Questions
M. alcicornis branching vs. encrusting vs. M. complanata Ruiz Ramos, Weil, Schizas (2014). Zoological Studies. M. complanata M. alcicornis branchingM. alcicornis encrusting MORPHOLOGY
Morphological Measurements g = Gastropore, d = Dactylopore, 1. Diameter of gastropore, 2. Diameter of dactylopore, 3. Distance among dactylopores, 4. Distances from gastropore to nearest dactylopore, 5. Distances among gastropores. Ruiz Ramos et al. (2014). Zoological Studies
GOOD BAD Ruiz Ramos et al. 2014. Zoological Studies
GOOD BAD GOOD Ruiz Ramos et al. 2014. Zoological Studies
Samples from 7 localities across the Caribbean were added for genetic analysis: Bocas del Toro (Panama), Grand Cayman, Mona (Puerto Rico - PR), Parguera (PR), Vieques (PR), Guadeloupe and Curaçao. One gene approach: mtCOI a variable marker in hydrozoans Molecular Methods
Unresolved Gene genealogy – COI gene RED = M. complanata BLUE = M. alcicornis (branching) GREEN = M. alcicornis (encrusting) Circle = M. striata Diamonds: haplotypes shared among the morphotypes. M. squarrosa We cannot distinguish M. complanata from M. alcicornis and M. striata. Millepora squarrosa is sufficiently different, genetically.
Study Design M. alcicornis branchingM. complanata Ion Proton (P1 chip) – One transcriptome per chip 2 transcriptomes M. alcicornis vs. 1 transcriptome M. complanata Test for consistency - 2 independent runs of M. alcicornis
Ion Proton Chip (P1 chip) Data M. alcicornis (run 1)M. alcicornis (run2) M. complanata (not analyzed yet) FASTQ Size (GB)5.913.512.3 Total Reads28,023,48064,001,281 Read Length~137 bp Read Length Range 4-2978-254 % GC50 After quality trimming Total Reads 24,527,97755,850,797 After removing Symbiodinium sp. FASTQ Size (GB) 5.2 GB11.5 GB % Reads Aligned to Symbiodinium 2.62%2.33% % of Reads Aligned to human 11.68 %11.10 % % of HQ Aligned Reads to human 5.35 %5.21 %
Pre-processing pipeline Symbiodinium and Human genomes RNA-seq data is aligned to reference genomes Contamination-free sequences
Assembly pipeline M. alcicornis RNA-seq data 1 M. alcicornis RNA-seq data 2
Bowtie (Aligner) – Aligns raw RNA-seq reads against assembled transcripts. Can be used to count the number of reads that are well represented in the assembled transcriptome. Can be used for visualization of reads alignment, information about read depth. RSEM – Also uses Bowtie to estimates gene and isoform expression levels from RNA-Seq data for each sample). Merge - Create a matrix using the expected fragment count data produced by RSEM across samples used for differential expression analysis. EdgeR - Estimates differential gene expression across samples (pairwise comparison). Post-Assembly Analysis Pipeline
Data from 253 putatively identified gene components suggests consistency between runs in Ion Proton Differential Expression between replicated data sets –M. alcicornis 224 (89%) 29 (11%)
Most abundant transcripts from the 2 runs are consistent TranscriptBLASTN functional annotation M. alcicornis #1 Normalized FPKM M. alcicornis #1 rank M. alcicornis #2 Normalized FPKM M. alcicornis #2 rank comp22892_c0 PREDICTED: Strongylocentrotus purpuratus uncharacterized LOC100892564, misc_RNA 1566319.9811689605.051 comp16419_c0 Millepora exaesa 18S ribosomal misc_RNA 287979.272422834.992 comp38876_c0 PREDICTED: Hydra magnipapillata hypothetical protein LOC100207766, transcript variant 2 258682.543209318.353 comp36161_c0 Bombyx mori gene for tRNAAsp 207115.614176415.555 comp22881_c0 Homo sapiens RNA, 45S preribosomal 5,ribosomal RNA 200387.665125102.459 comp38810_c0 Tubulipora flabellaris 28S ribosomal RNA gene, partialsequence 142164.766244234.793 comp37311_c0 Homo sapiens RNA, 45S preribosomal 5,ribosomal RNA 130956.657 6 comp39007_c0 Hydractinia symbiolongicarpus clone YU0089N10, complete sequence 111747.528144085.957 comp36078_c1 Millepora sp. EK2011 mitochondrion, partial 106730.59127298.328 comp39538_c1 Millepora sp. AMN2008 28S, partial sequence 98143.1410 comp39494_c0 Millepora sp. AMN2008 28S, partial sequence 89447.6710
Conclusions Consistency between the two Ion Proton runs on M. alcicornis Despite the high number of reads the resulting data sets were relatively small Lack of a reference genome/transcriptome We cannot explain the high contamination of rRNA (RiboMinus Kit–removes 12S and 18S rRNAs instead of the micropoly(A) Purist Kit which selects for mRNAs.
Continuing work… We are adding paired-end Illumina data for M. alcicornis and M. complanata. Generate high quality transcriptomes of M. striata and M. squarrosa. Perform transplant experiments and examine gene expression patterns in native vs. transplanted M. alcicornis colonies.
Acknowledgments We thank the Seven Bridges Genomics: Sebastian Wernicke, Lu Zhang Nemanja Ilic, and Jelena Radenkovic, for the bioinformatics analysis and guidance in this project. We thank the NIH Cancer Genome Facilities for allowing to use the Ion Proton and their facilities. The school of Arts and Sciences, University of Puerto Rico supported partially the travelling costs. Puerto Rico Sea Grant for providing funds for additional Illumina runs and training of students in NGS techniques