RNA-Seq transcriptome analysis of Gonium pectorale cell cycle

Dr. Tara N. MarriageKansas State University

Postdoc, Olson Lab

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 related unicellular and

multicellular species

~200 mya~ 1 bya

From Kirk, BioEssays 27 299-310 (2005)

Similarity in Genomes between Chalmydomonas and Volvox

Ferris, Olson et al. Science 328, 352 (2010)

From Kirk, BioEssays 27 299-310 (2005)

Similarity in Genomes between Chalmydomonas and VolvoxNumber of genes in Chlamydomonas: 14,516Number of genes in Volvox: 14,520Different by ~25 genes

Multicellularity NOT a major genomic innovation

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)

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:

Comparative transcriptomics of Gonium and Chlamydomonas cell

cycles

G. pectorale Genomic informationGenome sequencing a collaborative project

Generated via 454 sequencing

Draft genome:1781 scaffoldsN50 ~800kb

Merchant et al. Science 318 245 (2007)

Prochnik et al. Science 329 223 (2010)

G. pectorale Genomic informationGeneration 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

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 assemblyGenome annotation and comparisonRNA-Seq annotation and comparison

• Upload your scripts and enjoy!

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

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

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 unique barcode libraries

• Libraries sequenced on 1 lane of Illumina Hi-Seq2000

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

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

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

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

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 CycB1

4.92 CDKB1

1.99 CDKA1

3.87 MinE

4.45 pherophorin

4.03 metalloproteinase

1.88 CycD3

3.33 mot3

0

2

4

6

8

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12

14

16

wee1FP

KM

Pre-commitm

ent

Commitment

Post-co

mmitment

Mito

sis

Time point

0

0.5

1

1.5

2

2.5

3

SeparaseFP

KM

Pre-commitm

ent

Commitment

Post-co

mmitment

Mito

sis

Time point

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

0

1

2

3

4

5

6

7

8

9

CDKG1FP

KM

Pre-commitm

ent

Commitment

Post-co

mmitment

Mito

sis

Time point

0

20

40

60

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100

120

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160

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200

PherophorinFP

KM

Pre-commitm

ent

Commitment

Post-co

mmitment

Mito

sis

Time point

0

1

2

3

4

5

6

7

8

9

10

MetalloproteinaseFP

KM

Pre-commitm

ent

Commitment

Post-co

mmitment

Mito

sis

Time point

Other aspects to investigate

• Figure out clustering/heatmap

• Genes with dNdS ratios ≥ 1.0– Strong multicellularity gene candidates

• Cell cycle gene expression

Work in progress

Compare Gonium and Chlamydomonas cell cycle transcriptomes

Fine-scale Gonium cell-cycle transcriptome

Investigate non-coding portion of genomemiRNA transcriptome in GoniumChanges in UTRsTranscript rearrangements

Acknowledgements• Dr. Brad Olson• Olson Lab members

Chris BergerJaden AndersonSarah CosseyAndrea KiefferNicole Richardson

• COBRE (CMADP)• K-INBRE (NIH)• K-State Johnson Center

for Basic Cancer Research

• Collaborators Gonium pectorale Genome

Hisayoshi NozakiTakashi HamajiAtsushi ToyodaMasahiro SuzukiHiroko Kawai-ToyookaAsao Fujiyama

Olson lab http://www.k-state.edu/olsonlab/

• K-State Ecological Genomics Institute

• K-INBRE Bioinformatics Core at KSU