RNA-Seq transcriptome analysis of
Gonium pectorale cell cycle
Dr. Tara N. Marriage
Kansas State University
Postdoc, Olson ...
Multicellularity: A common evolutionary
occurrence
From Nicole King, Dev. Cell 7 313-25 (2004)
From Kirk, BioEssays 27 299-310 (2005)
Volvocine algae: Model system to study evolution of
multicellularity
Closely relate...
From Kirk, BioEssays 27 299-310 (2005)
Similarity in Genomes between Chalmydomonas and
Volvox
PRP4
RB
MAT3
EIF5Bb
UCP
PR46...
Unicellular vs. multicellular colonial life cycle
Chlamydomonas daughter cells separate after division yet Gonium
daughter...
Multiple fission and the cell cycle
Gp Cr
S Gp
Cr
Gp Cr
Gp
Cr
Mitosis
Pre-commitment
Commitment
Post-commitment
From Kirk, BioEssays 27 299-310 (2005)
Hypothesis
Changes in cell-cycle regulation result in
multicellularity
Approach:
Co...
G. pectorale Genomic information
Genome sequencing a
collaborative project
Generated via 454
sequencing
Draft genome:
1781...
G. pectorale Genomic information
Generation of gene models
• Informed gene prediction with
Augustus
• Functional annotatio...
Share your scripts on github!!
KINBRE-script-share
https://github.com/organizations/i5K-KINBRE-script-share/teams/451046
•...
Augustus gene prediction with RNA-seq data
Two-step iterative mapping approach
• RNAseq reads mapped to genome with Tophat...
Investigating the cell cycle transcriptome in Gonium
Cells collected from biological replicates at hourly time points
acro...
Pooling of RNA across time points
Pre-commitment
Commitment
Post-commitment
Mitosis
RNA library construction and
sequencing
• Libraries made for 4 time point RNA pools per
each biological replicate (3)---12...
Investigating the cell cycle transcriptome in Gonium
Cells collected from biological replicates at hourly time points
acro...
Read mapping and Differential gene
expression analysis
• Read mapping with Tophat v2.0.8
– Barcode libraries mapped indivi...
Read mapping and Differential gene
expression analysis
• Cuffmerge on cufflinks output
– Default parameters except provide...
Gonium transcriptome results
• Approximately 19 million high-quality 100bp
paired-end reads
• Mapping only to CDS, approxi...
Hierarchical clustering analysis: Preliminary results
Genes differentially expressed during
mitosis
Log 2 Fold change Gene/function
3.71 Separase
1.76 CDKG1
3.16 wee1
3.28 CycB...
0
2
4
6
8
10
12
14
16
FPKM
wee1
Time point
0
0.5
1
1.5
2
2.5
3
3.5
FPKM Separase
Time point
Genes differentially expressed during
mitosis
Log 2 Fold change Gene/function
3.71 Separase
1.76 CDKG1
3.16 wee1
3.28 CycB...
0
1
2
3
4
5
6
7
8
9
FPKM
CDKG1
Time point
0
50
100
150
200
250
FPKM
Pherophorin
Time point
0
1
2
3
4
5
6
7
8
9
10
FPKM
Metalloproteinase
Time point
Other aspects to investigate
• Figure out clustering/heatmap
• Genes with dNdS ratios ≥ 1.0
– Strong multicellularity gene...
Work in progress
Compare Gonium and Chlamydomonas cell cycle
transcriptomes
Fine-scale Gonium cell-cycle transcriptome
Inv...
Acknowledgements
• Dr. Brad Olson
• Olson Lab members
Chris Berger
Jaden Anderson
Sarah Cossey
Andrea Kieffer
Nicole Richa...
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RNA-Seq transcriptome analysis of Gonium pectorale cell cycle

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Tara Marriage KSU
RNA-Seq transcriptome analysis of Gonium pectorale cell cycle.
http://www.k-state.edu/olsonlab/people.html

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  • 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
  • RNA-Seq transcriptome analysis of Gonium pectorale cell cycle

    1. 1. RNA-Seq transcriptome analysis of Gonium pectorale cell cycle Dr. Tara N. Marriage Kansas State University Postdoc, Olson Lab
    2. 2. Multicellularity: A common evolutionary occurrence From Nicole King, Dev. Cell 7 313-25 (2004)
    3. 3. From Kirk, BioEssays 27 299-310 (2005) Volvocine algae: Model system to study evolution of multicellularity Closely related unicellular and multicellular species ~200 mya~ 1 bya
    4. 4. From Kirk, BioEssays 27 299-310 (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) Number of genes in Chlamydomonas: 14,516 Number of genes in Volvox: 14,520 Different by ~25 genes Multicellularity NOT a major genomic innovation
    5. 5. Unicellular vs. multicellular colonial life cycle Chlamydomonas daughter cells separate after division yet Gonium daughter cells remain attached. Cyclins/CDK’s (Cell cycle activator) MAT3/RB (Cell cycle repressor) E2F/DP (Transcription Factor, cell cycle activator)
    6. 6. Multiple fission and the cell cycle Gp Cr S Gp Cr Gp Cr Gp Cr Mitosis Pre-commitment Commitment Post-commitment
    7. 7. From Kirk, BioEssays 27 299-310 (2005) Hypothesis Changes in cell-cycle regulation result in multicellularity Approach: Comparative transcriptomics of Gonium and Chlamydomonas cell cycles
    8. 8. G. pectorale Genomic 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)
    9. 9. 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
    10. 10. 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!
    11. 11. 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
    12. 12. 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
    13. 13. Pooling of RNA across time points Pre-commitment Commitment Post-commitment Mitosis
    14. 14. 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
    15. 15. 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
    16. 16. Read mapping and Differential gene expression analysis • Read mapping with Tophat v2.0.8 – 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
    17. 17. 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
    18. 18. Gonium transcriptome results • 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
    19. 19. Hierarchical clustering analysis: Preliminary results
    20. 20. Genes differentially expressed during mitosis Log 2 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
    21. 21. 0 2 4 6 8 10 12 14 16 FPKM wee1 Time point
    22. 22. 0 0.5 1 1.5 2 2.5 3 3.5 FPKM Separase Time point
    23. 23. Genes differentially expressed during mitosis Log 2 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. 24. 0 1 2 3 4 5 6 7 8 9 FPKM CDKG1 Time point
    25. 25. 0 50 100 150 200 250 FPKM Pherophorin Time point
    26. 26. 0 1 2 3 4 5 6 7 8 9 10 FPKM Metalloproteinase Time point
    27. 27. Other aspects to investigate • Figure out clustering/heatmap • Genes with dNdS ratios ≥ 1.0 – Strong multicellularity gene candidates • Cell cycle gene expression
    28. 28. Work in progress Compare Gonium and Chlamydomonas cell cycle transcriptomes Fine-scale Gonium cell-cycle transcriptome Investigate non-coding portion of genome miRNA transcriptome in Gonium Changes in UTRs Transcript rearrangements
    29. 29. 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

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