Identification of transcript and genetic features which impact on gene

expression in filamentous fungi

Gwen Cowley, Alistair Darby, Mark Caddick
Functional and Comparative Genomics Institute of Integrative Biology
Abstract
High-throughput RNA sequencing generates a global view of the transcriptome and provides unprecedented insights into gene structure. Novel
sequencing techniques are being used to define gene architecture as well as relative gene expression levels. These approaches are being applied
and compared in two unique filamentous fungi; Aspergillus nidulans and Mycelipthora thermophila.

Genome Statistics
Aspergillus nidulans1
Genome sequenced 2005
~ 30 Mb
Eight chromosomes
>10,000 protein coding genes, ~8% characterised
Myceliapthora thermophila2
Genome sequenced 2011
~ 38 Mb
Seven chromosomes
>9,000 protein coding genes, >200 CAZy enzymes

Bioinformatics
Improved gene models will greatly assist biotechnological
exploitation of these and related organisms and extend the
utility of PacBio technology for improving RNA analysis. Highly
expressed genes across different growth conditions provide a
starting point for the analysis of differential regulation and
functional elements within regulatory regions.
Reference
AnnotaEons
(GFF)

Sequencing
Next generation sequencing, including Illumina and SoLID help
characterise genomes. Having been sequenced in 2005, A.
nidulans is being used to improve our understanding of
transcriptional regulation. 5' specific transcriptome sequencing
has resulted in over 14,000 transcription start sites (TSS) being
mapped across its genome. Short consensus sequences,
significantly enriched around TSS have been identified.
Promoter elements can be used in the development of synthetic
gene switches and robust expression systems.

Figure 1. RNA-seq of A. nidulans has been used to identify

transcription start sites through 5 enriched sequencing
libraries. Increasing our understanding of gene architecture
provides insights on functional elements within these regions.
Alternative splicing patterns such as intron retention and exon
skipping contribute to alternative splicing and increase the
diversity of the transcriptome and proteome.

Refere
nce
Alignm
Genom
Tag
ent e File
gene
(sam/
(Fasta)
Output
counts
bam)
les:
(plain
Tables
text)
& Plots
of DGE

Sequence
Reads
(Fasta)
Map (BowEe/
TopHat)

Count reads per

gene (htseq-count)

DGE analysis
(edgeR)

Downstream analysis
(GO Term enrichements
etc)

Figure 3. Pipeline of RNA seq analysis. Bioinformatics

analysis of transcriptome data is not yet an automated process.
Each of the steps can be adjusted and optimised to suit the
different libraries and sequencing platform.

Low Cellulase vs. High Cellulase

High Cellulase vs. High Cellulase &

Reduced Protease Activity

High Cellulase under minimal vs.

complete media

Figure 4. Differentially expressed genes. Gene expression

across growth conditions provides insight of the metabolic
versatility of an organism. Red points represent significantly
differentially expressed genes with p-value < 1%. Blue bars
indicate 2-fold change

Next Steps & Conclusion

Integrated analysis of sequencing experiments, including novel
PacBio tools for transcriptome sequencing, base-modification
detection and Iso-Seq analysis5 can be used to identify gene
architecture, regulatory elements and splice patterns and
improve genome annotations. Our aim is a better
Figure 2. Distribution of promoter enriched sequences. DNA understanding of the factors that influence gene expression and
motifs enriched upstream of TSS demonstrate a skewed
stability of recombinant proteins to facilitate new knowledge for
distribution in promoters, consistent with a them having
the
bioindustry
and
medicine.
functional roles3.
References
1. Galagan et al., 2005 Nature, 438:1105 2. Berka et al., 2011 Nature Biotech, 29:922 3. Sibthorp et al., 2013 BMC Genomics. 14:847 4. Haas & Zody, 2005 Nature Biotech, 28:423 5. Au et al., 2013 PNAS,