High-Throughput Sequencing to Identify the Effects of
Chemical and Environmental Stressors
Steve B.
1
Wiseman ,
Yuhe
1
He ,
Markus
1,2
Hecker ,
John. P
1,3,4
Giesy ,
1,2
Jones
Paul D.
1Toxicology
Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada
2School of Environment and Sustainability, University of Saskatchewan, Saskatoon, Saskatchewan
3Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
4Dept. of Biology and Chemistry, City University of Hong Kong, Hong Kong, SAR, PR China
INTRODUCTION
RESULTS 2 – EFFECTS OF OSPW ON ABUNDANCES OF TRANSCRIPTS
Open format nucleic acid sequencing technology, such as
Illumina’s RNAseq, afford the opportunity to perform large-scale
analysis of gene expression in species for which there is little or
no sequence information. The fathead minnow (Pimephales
promelas) is a popular small fish model. Several cDNA
microarray platforms have been developed for this species.
However, no study has used high-throughput sequencing to
determine transcriptional responses to environmental stressors.
Objectives: Use Illumina RNAseq to quantify transcriptional
responses in the livers of fathead minnows exposed to:
 Oil sands process affected water (OSPW). OSPW is generated
during the extraction of bitumen form oil sands deposits of Alberta,
Canada. It can be toxic to aquatic wildlife, but little is know
about the specific mechanisms of toxicity. RNAseq is ideally
suited to studying the effects of this complex mixture as
mechanisms of toxicity are unknown.
 Low temperature stress (4oC for 3 months). There are questions
about the potential toxicities of chemicals towards aquatic
organisms as they are forced to adapt to changing temperatures
due to climate change.
METHODS
Assembly of the Reference Transcriptome
Sources of RNA
• RNA isolated from the livers of 16 minnows exposed to different
chemical and environmental stressors.
Sequencing and De Novo Assembly
• Samples sequenced on an Illumina Genome Analyzer IIx (75-bp
single end reads), or an Illumina HiSeq™ 2000 (100-bp paired-end
reads). Sequencing was performed at the National Research
Council Plant Biotechnology Institute (Saskatoon, SK, Canada).
• Contigs were de novo assembled using CLC Genomics
Workbench v5.0. The minimum contig length was 200 bases.
Annotation of Reference Contigs
• Identities of contigs were determined using Blast2GO v2.5.0.
using a minimum E-value of 10-5.
 Rationale:
• OSPW is a complex mixture of water, silt, clay, inorganic, and organic compounds.
• Identities of most organic compounds in OSPW are not known.
• Greater than 109 m3 of OSPW is stored in tailings ponds.
• Naphthenic acids (NAs) thought to be the main drivers of the toxicity of OSPW.
• Because of the complexity of the mixture it was hypothesised that OSPW might
have multiple mechanisms of toxicity.
Downregulated
(78)
Table 2.2: Changes in abundances of
transcripts of xenobiotic metabolism.
 Study 2: Effect of long-term exposure to 4oC water on
abundances of transcripts in livers of fathead minnows.
• RNA was isolated from livers of fathead minnows exposed for 3
months to dechloroinated tap water at 20oC or 4oC.
• Samples were sequenced on an Illumina HiSeq™ 2000 using 100bp paired-end reads.
 RNAseq
• Reads were mapped to the reference transcriptome and abundance
of transcripts determined as RPKM (Reads Per Kilobase of
transcript per Million mapped reads).
• Data was quantile normalized and analyzed by Baggerley’s test
with a false discovery rate of 5%.
• A 2-step process was used to identify transcripts of greater or
lesser abundance:
 Identify transcripts that were statistically different than the
controls (corrected p-value < 0.05).
 Identify transcripts that were of 1.5-fold greater or lesser
abundance in the treatment relative to the control.
• Transcripts were annotated using gene ontology (GO) terms and
Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping
using Blast2GO v2.5.0.
RESULTS 1 – REFERENCE TRANSCRIPTOME
Table 1: Summary of RNAseq, de novo assembly,
and Blast2GO annotation.
Total Number of Reads
877,829,873
Number of De Novo Assembled Contigs
62,103
Min Contig Length
200 bp
Max Contig Length
20,818 bp
Mean Contig Length
1250 bp
N50*
2095 bp
Number of Contigs with E-value < 10-5
25,342
* N50 – length for which 50% of the sequences in the assembly is
in a contig of this length or greater.
Table 2.3: Changes in abundances of
transcripts of oxidative stress.
Fold Change
Transcript
2.1
Glutathione synthase
3.1
Cytochrome P4502k6
11.7
Glutathione peroxidase
1.7
Cytochrome P4502k1
10.1
Glutathione reductase
3.2
Cytochrome P4502J28
2.7
Thioredoxin
2.5
UDP-glucuronosyltransferase
2A3
Sulfotransferase 1-3
7.2
Thioredoxin reductase
2.7
Transketolase
2.4
6-Phosphogluconate
dehydrogenase
Glucose-6-phosphate
dehydrogenase
10.1
1.8
Glutathione-S-transferase
> 20.0
Multidrug resistance associated
protein 2
3.3
Table 2.4: Changes in abundances of
transcripts of apoptosis.
Transcript
Fold Change
Cytochrome P4501a
2.7
Apoptosis inducing
factor 3
Apoptosis inducing
factor M2
Poly (ADP-ribose)
polymerase-14
Damage-regulated
autophagy modulator
Programmed cell death
protein 4
FOX 03A
Fold Change
4.3
4.1
4.3
> 20.0
1.5
-3.3
• Constituents of OSPW might have by activated aryl-hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) or
pregnane-x-receptor (PXR) signaling, resulting in the expression of genes that encode proteins that metabolize xenobiotics.
• These proteins might metabolize organic constituents of OSPW, including NAs.
• Metabolism by P450s can lead to the production of reactive oxygen species, and cells respond by expression of antioxidant genes.
• Reactive oxygen species might stimulate caspase-independent apoptosis and autophagy.
RESULTS 3 – EFFECTS OF COLD ACCLIMATION ON ABUNDANCES OF TRANSCRIPTS
Table 3.1: Changes in abundances of
transcripts of cytochrome P450 enzymes.
Quantification of Gene Expression
 Study 1: Effects of OSPW on abundances of transcripts in
livers of sexually maturing male fathead minnows.
• RNA isolated from livers of 3 male minnows exposed to
dechlorinated tap water and 3 male minnows exposed to OSPW.
• Samples were sequenced on an Illumina HiSeq™ 2000 using 100bp paired-end reads.
Figure 1: Changes in abundances
of transcripts in livers of minnows
exposed to OSPW.
Upregulated
(109)
Transcript
Figure 2: Aerial view of an oil sands extraction facility
adjacent to the Athabasca river. Some freshwater is
drawn from the river for use in the extraction process. A
no-release policy requires that OSPW be stored and
cannot be released to the natural environment.
(image from nationalgeographic.com)
Downregulated
(986)
Upregulated
(1500)
Figure 2: Changes in hepatic transcript abundance
in fathead minnows exposed to 4oC for 3 months.
Table 3.2: Changes in abundances of
transcripts of glycolysis, gluceoneogenesis,
and Krebs cycle.
Transcript
Fold Change
Transcript
Fold Change
Cytochrome
P4502x
Cytochrome P4502j
-1.8
Cytochrome P450u
-2.5
Cytochrome P4503a
-6.6
Cytochrome P4504
-1.9
-5.5
Table 3.3: Changes in abundances of
transcripts of fatty acid synthesis.
Transcript
Fold Change
Glucokinase
> 50
Fatty acyl CoA synthase
15.4
Phosphoenolpyruvate
kinase
Triose phosphate
isomerase
Phosphoglucomutase
2.5
Acetyl CoA carboxylase
8.1
11.6
8.0
Stearoyl-CoA-9desaturase
Palmitoyl-CoA hydrolase
17.4
2.0
Phosphoglucokinase
1.8
Trans-2-enoyl-CoAreducatse
3-hydroxyacyl-CoAreductase
Malate dehydrogenase
3
Isocitrate dehydrogenase
1.5
Succinate dehydrogenase
2.2
4.8
4.1
Metabolism of xenobiotics and
endobiotics might be lesser in
fathead minnows exposed to 4oC
for 3 months.
Table 3.4: Changes in abundances of
transcripts of triglyceride metabolism.
Transcript
Fold Change
Phosphatidate
phosphatase
Glycerol kinase
4.2
12.4
Diacylglycerol kinase
3.7
Triacylglycerol
kinase
Diacylglycerol Oacyltransferase
6.0
3.2
• Aerobic metabolism of glucose might be greater in livers of fathead minnows exposed to cold water.
• Concentrations of triglycerides might be lesser in livers of minnows exposed to cold water. However, the elongation of fatty
acids and the synthesis of polyunsaturated fatty acids is greater in these minnows.
CONCLUSIONS
• De novo assembly of a transcriptome followed by RNAseq is effective for generating snapshots of transcriptional responses in
non-model species for which there is no or limited sequence information.
• RNAseq provides insight into mechanisms of toxicity and allows for the design of studies that elucidate potential cellular, tissue,
and organism level effects of exposure.
ACKNOWLEDGEMENTS
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Research grant from the Alberta Water Research Institute to J.P.G (Project # C4288)
Discovery grant from the Natural Science and Engineering Research Council of Canada (Project # 326415-07) and grants from Western Economic Diversification Canada (Project #
6578 and 6807) to J.P.G.
Research grant from the University of Saskatchewan VP Research to P.D.J.
Instrumentation grant from the Canada Foundation for Innovation to J.P.G.
The authors thank Warren Zubot of Syncrude Canada Inc. for supplying the OSPW.
Exposures were performed in the Aquatic Toxicology Research Facility at the University of Saskatchewan.