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19/01/2016
An Update on the Roslin Gene Expression
Dataset and Analysis
What Is A Gene Expression Atlas?
• Genome-wide map of gene expression.
• Transcripts are grouped according to their expression pattern.
• Determines co-expression and functional associations
between genes.
• Data can be used for gene annotation and creating/updating
gene models.
• As a searchable database an atlas is a critical resource to
complement genomic sequence data.
© N. Russell
Plant and Animal Genomes Conference Monday 11th Jan 2016
[email protected]
Why Study Gene Expression in Sheep?
Su et al. 2004 PNAS
Freeman et al. 2012 BMC Genomics
Harhay et al. 2010 Genome Biology
The Sheep Atlas Project
The Sheep Atlas Project has 3 key objectives:
• Enhancing desirable characteristics such as high quality wool
(e.g. Scottish Blackface) or large amounts of muscle (e.g.
Texel) can increase sheep value.
• Understanding gene expression and the genetic control of
complex traits will improve sheep breeding and the
management of disease.
• Sheep are a useful non-human model for studying the
fundamental biology of healthy adult mammals.
1. Analyse gene expression data across tissues and cell types in
sheep to provide insight into gene, cell and tissue function
throughout development.
2. Improve assembly and annotation of the recently released
sheep genome and increase the resources available for sheep
genetics.
Jiang et al. 2014 Science
3. Use this data to improve sheep productivity by improving our
understanding of the genetics of complex traits.
©N. Russell
The Starting Point - Collection of Tissues and
RNA Isolation
The Atlas Cross
Tissue Collections relevant for RNASeq and genome
sequencing:
6 Scottish Blackface x Texel Adults ~2 years of age
SCOTTISH BLACKFACE (BF)
TEXEL (T)
3 Blackface x Texel Lambs @ Birth
3 Blackface x Texel Lambs @ One Week
3 Blackface x Texel Lambs @ Eight Weeks
©N. Russell
3 Day 23 Blackface x Texel Embryos
3 pools of 8 Texel x ? Blastocysts (created using IVF)
www.texel.co.uk
Traits:
High Meat Quality
High Growth Rate
Increased Vigour
http://www.sheep101.info
Traits:
High Quality Wool
Hardiness
Survivability
40 tissues sequenced from adult sheep
(10 @ high depth >100 million reads, 30
@ >25 million reads).
10 GI tissues sequenced from each lamb
(all @ >25 million reads per sample)
©N. Russell
Whole genome sequencing on blood
samples from the 6 adults (10x coverage)
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RIN
Tissue Collection
Two Methods of Tissue Collection:
1.
•
2.
•
•
Preservation of tissues in RNAlater.
Slicing of tissues to <0.5cm diameter in any one direction
Snap freezing of problematic tissue.
E.g. brain and adipose
Slice tissues as for RNAlater to <0.5 diameter.
15 animals, 5 aliquots per tissue for an average of 40 tissues per
animal = 3000 samples!
Adult Tissue List
Skeletal Muscle longissimus dorsi
Heart Left Ventricle
Kidney Medulla
Kidney Cortex
Reticulum
Rumen
Omasum
Abomasum
Pylorus
Thoracic Oesophagus
Jejeunum
Ileum
Caecum
Colon (spiral)
Colon (distal)
Peyer's patch
Salivary Gland
Thyroid
Liver
Prescap lymph node
Popliteal lymph node
Testes
Ovary
Thymus
Lung parenchyma
Tonsil
Adrenal gland cortex
Adrenal gland medulla
Mesenteric Lymph Node
Spleen
Haemolymph node (iliac bifurcation)
Cerebellum
Pituitary gland
Hippocampus
Group
Musculoskeletal
Cardiovascular
Endocrine
Endocrine
GI tract
GI tract
GI tract
GI tract
GI tract
GI tract
GI tract
GI tract
GI tract
GI tract
GI tract
GI Tract
Immune
Endocrine
Endocrine
Lymphatic
Lymphatic
Reproductive
Reproductive
Immune
Respiratory
Immune
Endocrine
Endocrine
Lymphatic
Immune
Immune
Brain
Brain
Brain
Adult Male
Adult
Female
7.7
7.5
7
7.4
7.7
7.3
Total RNA - >100 million reads
7.6
7.1
per sample
7.8
7.7
7.1
8.1
- key subset of tissues to capture
7.7
7.6
8
7.5
global transcription
7.4
7
7.3
8.3
mRNA - >25 million reads per
7.2
7.2
sample
7.4
7.1
7.9
6.4
7.1
6.9
- particular tissues of interest
6.8
7.6
7.3
7.4
- mirrors the texel ‘mini’ atlas
7.2
8.3
7.7
8.1
(Jiang et al. 2014 Science)
7.8
7.5
7.2
7.6
7.9
8.3
8
N/A
N/A
8.3
7.6
8.7
7.1
7.2
8.3
8.5
7.7
9
7.7
8.7
7.5
8
7.4
7.3
8
8
7.4
7.7
8.5
7.8
7.6
8.5
+ bone marrow derived macrophages +/- LPS, blood leukocytes, PBMCs, alveolar macrophages and a subset of cardiovascular tissues
Lambs
Subset of 10 GI Tract
tissues from 9 lambs at
3 developmental stages
Data Processing Pipeline
RNA Seq Library Preparation
Genomic reads were aligned against the reference genome, Ovis aries v3.1 and
the variants called using GATK v3.4.46.
http://genomics.ed.ac.uk
Stranded Illumina TruSeq libraries (125bp paired-end) were generated by
Edinburgh Genomics on the HiSeq v2500 for tissues and cells.
Raw RNASeq reads are being processed using a HiSat2-Cuffmerge-Stringtie
pipeline with Oar v3.1 as the reference.
370 mRNA libraries (>25 million reads per sample) and 72 total RNA libraries (>100
million reads per sample) were generated in total.
On average 90% of reads passed initial QC and 65% mapped to the primary
alignment in a proper pair.
For 3 pools of 8 day 7 sheep blastocysts we used the Nugen Ovation® Single Cell
RNA-Seq System to generate libraries.
To estimate gene expression as FPKM (Fragments Per Kilobase of transcript per
Million mapped reads) we are using Ballgown and visualising the data in
Biolayout Express3D.
The genomes of the 6 adult atlas animals were sequenced at 10x coverage using
Tru-Seq Nano 350 gel free libraries (125bp paired-end) prepared from DNA
extracted from whole blood.
Preliminary Data Analysis
Genes in the sheep atlas libraries represented in the Ovis aries v3.1 reference
annotation.
Put node plot and gene expression graphs in here
% genes of this type
represented in sheep
No. genes in sheep
No. genes of this type present
No. genes of this type
% genes of this type
reference
in the sheep atlas but with no
atlas and with
present in the sheep
represented in the
Gene type
detectable expression
annotation (Oar
detectable expression (FPKM
atlas
sheep atlas
(FPKM >1) in at least
v3.1)
<1) in any tissue
one tissue
Mitochondrial rRNA
2
2
0
100
100
Mitochondrial tRNA
22
22
0
100
100
lincRNA
1858
919
469
49.46
24.22
miRNA
1305
1101
482
84.37
47.43
miscellaneous RNA
361
0
0
0
0
processed pseudogene
43
43
4
100
90.7
protein-coding
20921
17492
2200
83.61
73.09
pseudogene
247
240
78
97.17
65.59
rRNA
305
2
0
0.66
0.66
snRNA
1234
10
0
0.81
0.81
snoRNA
756
209
6
27.65
26.85
Total
27054
20040
3239
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Sheep Atlas Gene Expression Network Graph for a
Subset of Tissues in Biolayout Express3D
Adrenal Gland
Rumen
GI Tract
Cluster 13 – GI tract
Immune
Reproductive
Brain
Blastocysts
Reproductive
Blood/Immune
Heart/Skeletal Muscle
Immune
Cluster 11 – Skeletal Muscle
Using the Atlas Gene Expression Data
Identify candidate genes underlying phenotypic variation and
health.
Map the results across to other important ruminant species to
provide insight into ruminant divergence and disease resistance.
The variants called from the
whole genome sequencing
will be uploaded to EVA.
Make the data available via
genome browser software
and FAANG.
www.faang.org
Allelic Expression Imbalance
Allelic expression imbalance (AEI) is a phenomenon where one of the allelic transcripts is
overrepresented, relative to the other one in a gene transcript pool.
Cis-acting mutations may alter regulation for just one allele through a change to the
promoter/enhancer region.
The Callipyge phenotype which occurs in American Dorset lambs is an example of extreme AEI
known as ‘genomic-imprinting’ .
Improved understanding of the genetic control
of complex traits in ruminants will…
• Lead to improvements
in sheep breeding and
disease management.
• Enhance sheep
productivity.
© N. Russell
Provide a hugely valuable resource for researchers to tackle
numerous topics in ruminant immunity and productivity.
From Crowley et al. Nature Genetics 47, 353–360 (2015) doi:10.1038/ng.3222
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Acknowledgements
Iseabail Farquhar
Mary McCulloch
David Hume
Gemma Davis
Rachel Young
Lucas Lefevre
Clare Pridans
Rocio Rojo
Zofia Lisowski
Kristin Sauter
Lindsey Waddell
Mark Barnett
Sara Clohisey
Laura Glendinning
Yolanda Corripio-Miyar
Tim King
Alan Archibald
Heather Finlayson
Alison Wilson
Christine Burkhard
Alex Brown
Fiona Houston
Pip Beard
Erika Abbondati
Bruce Whitelaw
Chris Proudfoot
Simon Lillico
Zuyong He
Kim Summers
Ailsa Carlisle
Gwen Tsang
Photography
Norrie Russell
Dryden Farm Staff
Adrian Ritchie
Peter Tennant
John Bracken
Dougie McGavin
David Chisholm
Allison Mackenzie
Bioinformatics
Steve Bush
Edinburgh Genomics
Mick Watson
Richard Talbot
Helen Gunter
David Morrice
Thank you for listening!
http://www.scottish-blackface.co.uk
© N. Russell
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