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
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
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