RNA-Seq transcriptome analysis of Gonium pectorale cell cycle
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Transcript of RNA-Seq transcriptome analysis of Gonium pectorale cell cycle
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)
Number 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 [email protected]
• 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
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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
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60
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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
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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