Functional Genomics Overview - Bioinformatics-core-shared .

Functional Genomics OverviewRORY STARKPRINCIPAL BIOINFORMATICS ANALYSTCRUK – CAMBRIDGE INSTITUTE18 SEPTEMBER 2017

Agenda– What is Functional Genomics?– RNA Transcription/Gene Expression– Measuring Gene Expression Microarrays High-throughput Sequencing– Transcriptional Regulation Transcription factors Epigenetics Post-transcriptional regulation2

The Genome– Each cell contains a complete copy of the genome, distributedalong chromosomes (compressed and entwined DNA)– 3x109 (3Gb) base pairs in human DNA: 6 meters in each cell!– Encodes blueprint for all cellular structures and activities andwhich cells go where (somehow )3

FunctionalGenomics:Sequence vs.Function4

What accounts for the difference in phenotype?Different Genomes!

What accounts for the difference in phenotype?Different Functions!6

The Central Dogma of Molecular ionFunctionalGenomicsProteomics

So, what is functional genomics? Where sequence-based genomics looks at thestructure and components of genomes, andanalyses the similarities and differences betweengenomes Functional genomics looks at how genomes resultin cellular phenotypes, and analyses differences inhow the same genome functions differently indifferent cells, and how changes in genomes alterfunction8

GeneExpression(RNATranscripts)9

Gene expression experiments– Measure the expression levels of manygenes in parallel– Ideally, we’d measure all protein levels– However, proteomics is difficult!– Instead, use mRNA (“transcript”) levels asa proxy for protein levels– (How good a proxy is RNA?)– Several good ways to measure RNA– Analyses: Expression levels Differences in expression levels (DE) Patterns of expression Splicing and isoforms10mRNA vs Protein levels in YeastR 0.44Ghaemmaghami et al Nature 2003

What kinds of samples are we interested in?– Different tissues, same organism human brain/human liver– Same tissue, different organism human liver/mouse liver wt/ko– Same tissue, same organism, different condition benign/tumour treated/untreated– Time course (effect of treatment over time)– In vivo vs In vitro– 11

MeasuringGeneExpression12

Reverse transcription(mRNA - cDNA)– Most RNA-seq involveslarge populations of cells(106-7)– Most RNA-seq involvessequencing cDNAsynthesized using reversetranscription– Most RNA-seq involvessignificant amplification ofcDNA molecules via PCR13

Measuring cDNA: MicroarraysUse hybridization to measure abundance of mRNA transcriptsFix “probes” to a solid supportHybridize labeled samples of mRNA to probesUse labels to measure hybridization intensityRafa Irizarry1421

Microarrays: Scanninghttp://www.wormbook.org/chapters/www ly less than 1 inch width, spot diameter ! 0.1 mm18

Measuring cDNA: RNA-seq– High-throughput sequencingallows us to sequence arepresentative sample of thecDNA population “directly”– Each sequence “read” isaligned back to a referencegenome/transcriptome to seewhere it was transcribed from– We can count how manytranscripts came from eachgene16

Trends in Transcriptomics– Single-cell sequencing– Nanopore Sequencing Full-length transcript sequencing Direct RNA sequencing17

Beyond GeneExpression:TranscriptionalRegulation18

Transcriptional meAmino AcidsHKDESTNQCUGPAVILMFYW

Transcriptional regulatoryelementsGene/ProteinInteractionsDNA ssionSmall regulatoryRNA(eg microRNA)

Regulatory elements of interest include TRANSCRIPTION FACTORS– ChIPHISTONE MARKS– ChIPDNA METHYLATION– RRBS– MeDIPOPEN CHROMATIN– DNase Hypersensitivity– ATACÇHROMATIN STRUCTURE– HiCRNA POLYMERASE– Pol II ChIP

Cell differentiation22

And Beyond – Post-transcriptionalRegulation– Translational Efficiency– DATA INTEGRATION23

AcknowledgementsFormer lecturers whocontributed to theselecture notes:Benilton S CarvalhoNuno Barbosa-MoraisStephen J EglanNatalie Thorne

So, what is functional genomics? Where sequence-based genomics looks at the structure and components of genomes, and analyses the similarities and differences between genomes Functional genomics looks at how genomes result in cellular phenotypes , and analyses di