RNA-Seq Analysis of Gonium pectorale Cell Cycle Transcriptome
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This document summarizes Dr. Tara N. Marriage's RNA-Seq analysis of the cell cycle transcriptome in the multicellular alga Gonium pectorale. RNA was extracted from G. pectorale cells collected hourly across a 24 hour period and pooled into time points corresponding to different cell cycle phases. RNA-Seq libraries were constructed and sequenced, and the reads were mapped and analyzed for differential gene expression. Preliminary results identified over 2400 differentially expressed genes across the cell cycle and hierarchical clustering of expression profiles. Several key cell cycle genes were found to be differentially expressed during mitosis. The analysis is ongoing to further investigate cell cycle regulation and changes contributing to multicellularity in Gonium compared
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RNA-Seq Analysis of Gonium pectorale Cell Cycle Transcriptome
- 1. RNA-Seqtranscriptome analysis of Gonium pectorale cell cycle Dr. Tara N. Marriage Kansas State University Postdoc, Olson Lab
- 2. Multicellularity: A common evolutionary occurrence From Nicole King, Dev. Cell 7 313-25 (2004)
- 3. From Kirk, BioEssays27 299-310 (2005) Volvocine algae: Model system to study evolution of multicellularity Closely related unicellular and multicellular species ~200 mya~ 1 bya
- 4. From Kirk, BioEssays27 299-310 (2005) Similarity in Genomes between Chalmydomonas and Volvox PRP4 RB MAT3 EIF5Bb UCP PR46a THI10 NMDA1 GP2 ATPC EFG3 TwosyntenicblocksonScaffold2 1cM5cM0cM0cM0cM32.4cM 500 kb RB MAT3 Volvox female Linkage Group I Chlamydomonas Chromosome 6 MT + MTF Ferris, Olson et al. Science 328, 352 (2010)
- 5. From Kirk, BioEssays27 299-310 (2005) Similarity in Genomes between Chalmydomonas and Volvox Number of genes in Chlamydomonas: 14,516 Number of genes in Volvox: 14,520 Different by ~25 genes Multicellularity NOT a major genomic innovation
- 6. Unicellular vs. multicellular colonial life cycle Chlamydomonas daughter cells separate after division yet Goniumdaughter cells remain attached. Cyclins/CDK’s (Cell cycle activator) MAT3/RB (Cell cycle repressor) E2F/DP (Transcription Factor, cell cycle activator)
- 7. Multiple fission and the cell cycle Gp Cr S Gp Cr Gp Cr Gp Cr Mitosis Pre-commitment Commitment Post-commitment
- 8. From Kirk, BioEssays27 299-310 (2005) Hypothesis Changes in cell-cycle regulation result in multicellularity Approach: Comparative transcriptomics of Gonium and Chlamydomonas cell cycles
- 9. G. pectoraleGenomic information Genome sequencing a collaborative project Generated via 454 sequencing Draft genome: 1781 scaffolds N50 ~800kb Merchant et al. Science 318 245 (2007) Prochnik et al. Science 329 223 (2010)
- 10. G. pectorale Genomic information Generation of gene models • Informed gene prediction with Augustus • Functional annotations with blast2go • Orthologous groups with OrthoMCL, dual reciprocal blast • High-quality gene annotations and gene orthologs
- 11. Share your scripts on github!! KINBRE-script-share https://github.com/organizations/i5K-KINBRE-script-share/teams/451046 • Join the group e-mail kstate.bioinformatics@gmail.com • Create a folder for your lab in one of the three repositories: Transcriptome and genome assembly Genome annotation and comparison RNA-Seq annotation and comparison • Upload your scripts and enjoy!
- 12. Augustus gene prediction with RNA-seq data Two-step iterative mapping approach • RNAseq reads mapped to genome with Tophat – Alignment filtered with perl scripts to make intron hints file – Intron hints file fed to Augustus to generate exon-exon junction database • RNAseq reads mapped to exon-exon junction database with Bowtie – Mapped files were merged and filtered to make final intron hints file – Intron hints file again fed to Augustus to obtain gene predictions http://bioinf.uni-greifswald.de/bioinf/wiki/pmwiki.php?n=IncorporatingRNAseq.Tophat
- 13. Investigating the cell cycle transcriptome in Gonium Cells collected from biological replicates at hourly time points across 24 hour period Workflow RNA extracted and pooled based on cell cycle time points Construction of RNA libraries and Illumina Hi-Seq Alignment of reads and differential gene expression analysis
- 14. Pooling of RNA across time points Pre-commitment Commitment Post-commitment Mitosis
- 15. RNA library construction and sequencing • Libraries made for 4 time point RNA pools per each biological replicate (3)---12 unique barcode libraries • Libraries sequenced on 1 lane of Illumina Hi- Seq2000
- 16. Investigating the cell cycle transcriptome in Gonium Cells collected from biological replicates at hourly time points across 24 hour period Workflow RNA extracted and pooled based on cell cycle time points Construction of RNA libraries and Illumina Hi-Seq Alignment of reads and differential gene expression analysis
- 17. Read mapping and Differential gene expression analysis • Read mapping with Tophatv2.0.8 – Barcode libraries mapped individually – Default parameters except provided CDS Augustus gff3 file and genome fastq file • Transcript assembly with Cufflinks v2.0.2 – Default parameters except provided CDS Augustus gff3 file – Separate assembly for all barcode transcripts
- 18. Read mapping and Differential gene expression analysis • Cuffmerge on cufflinks output – Default parameters except provided CDS Augustus gff3 file • Cuffdiff for DGE – Merged gtf file from cuffmerge – Biological replicate SAM files supplied as comma separated list – Default parameters
- 19. Gonium transcriptomeresults • Approximately 19 million high-quality 100bp paired-end reads • Mapping only to CDS, approximately 35% of reads mapped • Over 2400 significantly differentially expressed genes across cell cycle
- 20. Hierarchical clustering analysis: Preliminary results
- 21. Genes differentially expressed during mitosis Log2 Fold change Gene/function 3.71 Separase 1.76 CDKG1 3.16 wee1 3.28 CycB1 4.92 CDKB1 1.99 CDKA1 3.87 MinE 4.45 pherophorin 4.03 metalloproteinase 1.88 CycD3 3.33 mot3
- 24. Genes differentially expressed during mitosis Log2 Fold change Gene/function 3.71 Separase 1.76 CDKG1 3.16 wee1 3.28 CycB1 4.92 CDKB1 1.99 CDKA1 3.87 MinE 4.45 pherophorin 4.03 metalloproteinase 1.88 CycD3 3.33 mot3
- 28. Other aspects to investigate • Figure out clustering/heatmap • Genes with dNdS ratios ≥ 1.0 – Strong multicellularity gene candidates • Cell cycle gene expression
- 29. Work in progress Compare Gonium and Chlamydomonas cell cycle transcriptomes Fine-scale Gonium cell-cycle transcriptome Investigate non-coding portion of genome miRNAtranscriptome in Gonium Changes in UTRs Transcript rearrangements
- 30. Acknowledgements • Dr. Brad Olson • Olson Lab members Chris Berger Jaden Anderson Sarah Cossey Andrea Kieffer Nicole Richardson • COBRE (CMADP) • K-INBRE (NIH) • K-State Johnson Center for Basic Cancer Research • Collaborators Gonium pectorale Genome Hisayoshi Nozaki Takashi Hamaji Atsushi Toyoda Masahiro Suzuki Hiroko Kawai-Toyooka Asao Fujiyama Olson lab http://www.k-state.edu/olsonlab/ • K-State Ecological Genomics Institute • K-INBRE Bioinformatics Core at KSU
Editor's Notes
- closely related group of species that span from unicellular, to colonial multicellular to multicellularity
- Cell cycle of chlamydomonas growth and division decoupled—cells go through period of growth (G1) and then undergo division (S/M) phaseGrowth occurs while light is availableDivison occurs in the darkSynchronycommitment
- Possibly expand this slide as well, at least for Augustus gene prediction with RNAseq data