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
TIGR Autoannotation vs Sanger Curated Annotation StatusCount Total Sanger genes analyzed360 Same gene structure137 Different gene structure177 Sanger missing in TIGR annotation37 Sanger matches multiple TIGR annotations2 Sanger, TIGR annotations opposite strands7 TIGR missing in Sanger annotation12 TIGR matches multiple Sanger annotations9
Using Ortholog Clusters to Identify Potential Annotation Problems
Using Ortholog Clusters to Identify Potential Annotation Problems Different exon number due to annotation discrepancy
We need to be able to distinguish annotation inconsistencies from real, interesting phenomena In some cases, differences in exon number are real
Expression profiling analysis to study Pathogenesis Response to fungicidal drugs Temperature-dependent gene expression - A. fumigatus is an environmental species can grow at temperatures as high as 55ºC can survive at temperatures up to 70ºC. - It is commonly isolated from metabolically heated compost heaps
The Beast: Microarray Robot from Intelligent Automation
Microarray data analysis Software freely available at, http://www.tigr.org/software Reference sample Query sample Hybridization Obtain signal intensity values from images Scanning Multi-experiment comparison Data Normalization and analysis
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 Mays Lab
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.
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.
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.
Dispersed in the genome Transposons in A. fumigatus
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_pctidavg_coveragenum_cogs 3 member+++70%86%5899 ++ 65%84%967 2 member+ +61%79%533 ++61%80%936 Species #genes included in COG percent of predicted proteome A. fumigatus750779% A. nidulans742975% A. Oryzae798857% Total22924 68%(22924/33552) Overview – comparative statistics
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
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
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
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
A. fumigatus vs. A. fischerianus Secondary 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
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
Aspergillus fumigatus AF293 David Denning Michael Anderson Arnab Pain Goeff Robson Javier Arroyo Goeff Turner David Archer Joan Bennett Matt Berriman Jean Paul Latge Paul Dyer Paul Bowyer Neil Hall Aspergillus nidulans – James Galagan Aspergillus oryzae – Masayuki Machida
TIGR Sequencing and Closure Tamara Feldblyum Hoda Khouri Annotation Jennifer Wortman Jiaqi Huang Resham Kulkarni Natalie Fedrova Claire Fraser Microarray H. Stanley Kim Dan Chen NIAID and Dennis Dixon