1. Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters
Dallery et al. 2017

2. Colletotrichum higginsianum
Max Planck Institute
Pathogenic fungus
Affects brassica crops, such as Arabidopsis thaliana, in tropical and subtropical regions
Important model pathosystem for looking at molecular basis of fungal pathogenicity and host response
O’Connell et al. 2012

3. Rationale Affects crop yields
Previous genome assembly was highly fragmented
Looking for role of transposable elements (TEs) in gene and genome evolution
Better understanding of genome structure of pathogenic fungi

4. Methods 10 μg genomic DNA → ~20kb size-selected library
Sequenced on PacBio RS II platform
De novo assembly with the Hierarchical Genome Assembly Process (HGAP) approach
Reads filtered for min. 500bp length
Genome consensus sequence polished with Quiver
Assembly validated w/ PCR
Illumina sequencing w/ 100 bp paired-end reads
Used only to detect sequence polymorphisms

5. Even More Methods
REPET pipelines to detect and classify TEs and simple sequence repeats
Analysis of repeat-induced point mutations
Gene predictions with MAKER2, SNAP, Augustus from Illumina reads
Functional annotations via BLASTp and Blast2GO and predictions from SMURF, antiSMASH v.3.0, SMIPS, and CASSIS
Phylogenetic analysis of secondary metabolism key genes (MEGA6 and Treedyn)

6. Final Methods Slide
Analysis of distance of TEs to genes and gene clusters
Segmental duplication analysis (SDDetector w/ PacBio unitigs)
Transcriptome analysis (previous RNA-Seq data)
Basically a bunch of experimental validation of transcriptome data

7. Genome Assembly 7.8 Gb of raw sequence reads
92,834 error-corrected reads
N50 length 16,193 bp
Final edited assembly =
28 unitigs (unitigs = high confidence contigs)
12 largest unitigs = chromosomes, 99.14% genome assembly
Total length = Mb
Not actually gapless = gap on Chr 7 (liars)

8. Genome Assembly Genome assembly compared to previous 2009 assembly
Assembly Statistics
2012 Assembly
2017 Assembly
PacBio read coverage _
133x
Sanger read coverage
0.2x
Illumina read coverage
76x
454 read coverage
25x
Genome physical size
53.35 Mb
Assembly length
49.05 Mb
50.72 Mb
Alignable sequence
77.14 kb
50.38 Mb
Number of contigs
10, 259 28
Largest contig
49.23 kb
6.04 Mb
N50 contig length
6.15 kb
5.20 Mb
Complete genes
2946 (79%)
3616 (97%)

9. Results 2699 MAKER2 genes match to previous gene models
2289 new genes w/ no match in previous annotation
Includes 132/133 genes on Chr 12

10. Results
Mini chromosomes 11 & 12 have half the gene content of the ‘core’ chromosomes
Lower gene expression
Much higher TE content

11. Results

12. Results
Secondary Metabolite (SM) Gene Clusters

13. Results
Genes in SM clusters + genes encoding candidate secreted effector proteins were found significantly closer to TEs than random genes over whole genome
Many copies of large TEs

14. Results Found 6 segmental duplications
4 of these are at chrom ends and/or regions of highly similar repeats

15. Actually Cool Results
Some TE families subject to Repeat-Induced Point (RIP) mutations
Occurs during meiosis (sexual reproduction)
This fungus is asexual
RIP occurred either during ancestral sexual state or there is cryptic meiosis happening
~30% TEs appear active
60% of expressed SM clusters only during plant infection

16. Conclusions
A complete genome assembly is key to analysis of TEs, teleomeres, structural rearrangements, and large gene clusters
The mini-chromosomes differ dramatically from the core genome in gene and repeat content
Resemble conditionally dispensable chroms.
Pathogenicity-related (?) genes
Repeat-mediated segmental duplication likely accelerated the pathogenicity-related gene evolution, e.g. ectopic recombination
SM gene cluster inventory will help to ID novel bioactive molecules and their biosythetic pathways

17. Questions
Would the Illumina library have helped the genome assembly if it had been included?
Are unitigs as good as scaffolds when used in their place?
If the fungus lost its mini-chromosomes, would it be significantly less pathogenic?