slide 1 · ppt file · web view2017-05-03 · what the aspergillus genomes have told us william c....
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What The Aspergillus Genomes Have Told Us
William C. NiermanThe Institute for Genomic Research
Rockville, MD
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Electrophoretic Karyotyping 5 day run
CHEF DRII 1.2% CGA, 1x TAE, 14C, 1.8 V/cm: 2200 s, 48 h; 2200-1800 s, 68 h sizes in Mb
5.74.6
3.5Sc Sp
5.01x 4.0
3.5
1.8Af
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Aspergillus fumigatus karyotype
1,789 Kb
3,779 Kb
2,021 Kb
3,992 Kb
4,018 Kb
4,834 Kb
4,891 Kb
3,933* Kb
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Optical Analysis
• Molecule maps generated from images of single DNA molecule digested with NheI
• Resolution (avg fragment size) 8.28kb• Total coverage: 8,987 Mbase, or 300x• Total of 8 chromosomes• Total size: 29.189 Megabases
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A. fumigatus chr5-7 contig placement
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Aspergillus fumigatus Chromosomes
Presumed centromeric area
Telomere
3
2
6
5
8
7
1
4
Mitochondrion
rRNA
32 Kb
4.9 Mb
4.8 Mb
4.0 Mb
3.9 Mb
3.9 Mb
3.6 Mb
2.0 Mb
1.8 Mb
2.2 2.7
1.8 3.0
1.3 2.8
2.50.4 0.70.3
1.2 2.6
1.3 2.5
0.7 1.3
0.8 1.0
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Nuclear Genome
Size (Mb) 29.2
GC Content 49.9%
Gene Number 10,034
Mean Gene Length (bp) 1,431
Percent Coding 50.1
Percent Genes with Introns 77.0
Mitochondrial Genome
Size (bp) 31,892
GC Content 25.41%
Gene Number 16
Mean Gene Length (bp) 1,189
Percent Coding 44.1%
Percent Genes with Introns 6.2
tRNA Number 10
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Synteny Map of A. fumigatus, A. nidulans, A. oryzae
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Aspergillus fumigatus Synteny AF293 vs CEA10
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TIGR Autoannotation vs Sanger Curated Annotation
• Status Count• Total Sanger genes analyzed 360• Same gene structure 137• Different gene structure 177• Sanger missing in TIGR annotation 37• Sanger matches multiple TIGR annotations 2• Sanger, TIGR annotations opposite strands 7• TIGR missing in Sanger annotation 12• TIGR matches multiple Sanger annotations 9
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Using Ortholog Clusters to Identify Potential Annotation Problems
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Using Ortholog Clusters to Identify Potential Annotation Problems
Different exon number due to annotation discrepancy
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We need to be able to distinguish annotation inconsistencies from real, interesting phenomena
In some cases, differences in exon number are real
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Expression profiling analysis to study
• Pathogenesis• Response to fungicidal drugs• Temperature-dependent gene expression
- A. fumigatus is an environmental speciescan grow at temperatures as high as 55ºC can survive at temperatures up to 70ºC.- It is commonly isolated from metabolically heated compost heaps
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The Beast: Microarray Robot from Intelligent Automation <http://www.ias.com>
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Microarray data analysisSoftware freely available at, < http://www.tigr.org/software >
Referencesample
Querysample
Hybridization
Obtain signal intensity values
from images
Scanning
Multi-experiment comparison
DataNormalizationand analysis
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Example hybs(A flip-dye set)
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Temperature shift experiments
• Two shift experiments– 30ºC to 37ºC shift– 30ºC to 48ºC shift
• Design– A. fumigatus was grown in a rich medium at 30ºC
for two days from conidia, and shifted to 37ºC or to 48ºC.
– Samples were taken throughout a time course.• Samples were prepared in Greg May’s Lab
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1. A number of genes of various functional roles express differentially at each temperature.
2. More genes are shifted to down-regulation than up-regulation at 48˚C in comparison to 30˚C.
3. More genes are turned up at 37˚C when temperature was shifted from 30˚C.
This suggests that the fungus has more variety of activities at 37˚C than does at the other temperatures, and it is least active at 48˚C.
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30ºC to 37ºC784 genes
heat shock proteins
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30ºC to 48ºC257 genes
heat shock proteins
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Putative virulence genes
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1. More heat shock and stress-responsive genes (ex. those coding for heat shock proteins and chaperons) are highly expressed at 48˚C than are at lower temperatures, indicating that the fungus is under heat stress.
2. More putative virulence genes (ex. those coding for the proteins responsive to oxidative stress and host immune system and for toxin production) are highly expressed at 37˚C, although there is no contact with host cells.
While predicted function from each gene should be experimentally verified, we suggest from this study that temperature is a key environmental signal for the organism that triggers gene regulation cascades that may ultimately lead to adaptation to a specific new environment.
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Many transposases, especially those of Mariner-4 type, are highly expressed at 48˚C.
It will be interesting to see if the high expression of the transposases actually leads to the transposition events of the transposons.
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0
5
10
15
20
25
Dispersed in the genome
Transposons in A. fumigatus
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The ortholog was computed by performing an all vs. all BlastP of the three
proteomes with a cut-off of 1 x e-15 (no length requirement). The mutual best
hits were then organized into clusters based on shared protein nodes.
COGA.
fumigatusA.
OryzaeA.
nidulansavg_pcti
davg_coverag
enum_cog
s
3 member + + + 70% 86% 5899
+ + 65% 84% 967
2 member + + 61% 79% 533
+ + 61% 80% 936
Species#genes included in
COGpercent of predicted
proteome
A. fumigatus 7507 79%
A. nidulans 7429 75%
A. Oryzae 7988 57%
Total 22924 68%(22924/33552)
Overview – comparative statistics
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Aspergillus fumigatus Unique Genes
• Vast majority are hypothetical• Includes
– Several transcriptional regulators– A chaperonin– An hsp 70 related protein– ArsC, arsenate reductase– Teichoic Acid Biosynthetic Protein
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Comparative Genomic Hybridization (CGH)
• Competitive hybridization between two genomic DNA
• Uses microarray to score the presence of genes relative to the reference on the microarray
• Provides a quick and easy way of comparing the gene content of a reference organism relative to an unsequenced CLOSE relative
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A. fumigatus vs. A. fischerianus
• Within same cluster by large subunit rRNA analysis
• Average DNA identity of ~ 90% based on 4X contigs of A. fischerianus
• A. fischerianus rarely identified as a pathogen
• A. fischerianus possesses a known sexual cycle
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A. fumigatus vs. A. fischerianus
• Relative to A. fumigatus, A. fischerianus is missing 700 genes – 13 Secondary metabolite genes– 28 Transcription regulators and protein
kinases– 21 Transporters– 199 Metabolic and other proteins– 400 Hypothetical proteins
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CGH between A. fumigatus and A. fischerianus
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A. fumigatus vs. A. fischerianusSecondary Metabolite Gene Summary
• Relative to A. fumigatus, A. fischerianus is missing • 3 of 7 DMAT genes
• 6 of 14 PKS genes
• 1 of 15 NRPS genes
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Additional related genomic projects underway or soon to be initiated
• Comparative analysis of Aspergillus fumigatus AF293 and CEA10
• Sequencing of Aspergillus flavus• Sequencing of Aspergillus terreus• Sequencing of Aspergillus clavatus• Sequencing of Aspergillus fischerianus• CGH of Neosartorya fennelliae with A. fumigatus• CGH across multiple A. fumigatus strains
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Aspergillus fumigatus AF293
David DenningMichael AndersonArnab PainGoeff RobsonJavier ArroyoGoeff TurnerDavid Archer
Joan BennettMatt BerrimanJean Paul LatgePaul DyerPaul BowyerNeil Hall
Aspergillus nidulans – James GalaganAspergillus oryzae – Masayuki Machida
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TIGR
Sequencing and ClosureTamara FeldblyumHoda Khouri
AnnotationJennifer WortmanJiaqi HuangResham KulkarniNatalie Fedrova
Claire Fraser
MicroarrayH. Stanley KimDan Chen
NIAID and Dennis Dixon