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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) 1 19/01/2016 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 2 19/01/2016 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 3 19/01/2016 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 4