NHLBI NHLBI Genomics Core FacilityGenomics Core Facility

Mark Gladwin M.DMark Gladwin M.DEric Billings, Ph.DEric Billings, Ph.D

Robert Balaban, Ph.DRobert Balaban, Ph.DSteering Committee?Steering Committee?

Kim Woodhouse

Hangxia Qiu, Ph.D

Tony Cooper Xiuli Xu, Ph.D

Bio-InformaticsBio-Informatics

Nalini Raghavachari, Ph.D

Wet labWet lab

Peter Munson, Ph.DPeter Munson, Ph.DDirector, MSCL, CITDirector, MSCL, CIT

Delong Liu, Ph.DDelong Liu, Ph.D

MSCL Staff, CITMSCL Staff, CITJim Taylor, M.D

New Organizational Structure

Primary GoalsPrimary Goals

• To provide investigators with high quality, To provide investigators with high quality, state-of-the-art gene expression profiling & state-of-the-art gene expression profiling & genotyping services in a timely fashion using genotyping services in a timely fashion using the affymetrix platformthe affymetrix platform

rigorous standardization of protocols and rigorous standardization of protocols and multiple quality control checksmultiple quality control checks

• To provide streamlined data analysis and To provide streamlined data analysis and identify signature genesidentify signature genes

Application of complex statistical toolsApplication of complex statistical tools

Resources in the Core

Nanodrop spectrophotometer

Agilent’s Lab on a chip

GCAS Robot for sample processing

Peg ArraysHuman U133A gene chips only

Hyb Oven Fluidic Station Scanner

Scanner

ABI 7900 – Sequence Detection SystemABI 7900 – Sequence Detection SystemQ-PCRQ-PCR

Perkin Elmer ScanArray 5000XL

2 color spotted arrays

Application software in the coreApplication software in the core

• GCOSGCOS - Data collection- Data collection

• MSCL ToolboxMSCL Toolbox - Normalization/Analysis- Normalization/Analysis

• JmpJmp - Data Set Analysis- Data Set Analysis

• GenespringGenespring - Data Set Analysis- Data Set Analysis

• Partek ProPartek Pro - - Normalization/AnalysisNormalization/Analysis

• IngenuityIngenuity - Pathway analysis- Pathway analysis

• MetacoreMetacore - Pathway Analysis- Pathway Analysis

Expression Profiling WorkflowExpression Profiling Workflow

2 µg

(Spike in labeling controls)

(Spike in hyb controls)

250 ng Genomic DNA Restriction Digestion

Nsp1 Nsp1Nsp1

Single Primer Amplification

Fragmentation and Labeling

Hyb, Wash, and Stain. Scan and Data Analysis

200 – 1100 bases

Nsp1

Genotyping WorkflowGenotyping Workflow

Day 1

Day 2 Day 3

Backup servers

Scanner

NIHLIMS

GCOS Client Signet Investigator

1 2 3

DVD archives of data

Inage and QC data are checkedRetrieval of data from

NIHLIMS

Normalize & Transform dataPrincipal Component Analysis

Statistical Tests - t-tests, ANOVA, Cluster

analysis

Multiple comparison corrections

Apply FDR and FC filters select gene lists

Gene Ontology, Pathway analysis, Bio-Informatics

Data Flow and AnalysisData Flow and Analysis

Online access to project dataOnline access to project data

Basic Level of ServiceBasic Level of Service

Consultation on project

Recommendations on RNA isolation

Amplification of RNA ~ 3000cells/3-5 ng of RNA

Simple statistical analysis, gene

selection

Pathway Analysis(GSEA, Genego, GoScan, GoMap)

Standard LevelStandard Level

Target(s) identification

Help with Publication

Collaborate on experimental design

RNA isolation

Amplification of RNA

Novel / Complex Statistical Methods(Munson’s group)

Novel Pathway Analysis

Target(s) identification

Taqman Analysis (qPCR)

Help with Publications

Advanced AnalysisAdvanced Analysis

co-authorship in publications

New Service InitiativesNew Service Initiatives

• Taqman Low Density Arrays (can analyze 8 samples for Taqman Low Density Arrays (can analyze 8 samples for 24 different genes)24 different genes)

• Exon and Tiling Array MappingExon and Tiling Array Mapping

• Method development for the isolation and analysis of Method development for the isolation and analysis of picogram amounts of RNA in collaboration with bio-tech picogram amounts of RNA in collaboration with bio-tech companiescompanies

• Service for non NHLBI investigators at a nominal costService for non NHLBI investigators at a nominal cost

Core PoliciesCore Policies• Investigator has to provide chips and samples to the core (We need Investigator has to provide chips and samples to the core (We need

at least 2at least 2µg total RNA in 4µl volume, 250ng genomic DNA)µg total RNA in 4µl volume, 250ng genomic DNA)

• Submitted RNA/DNA samples should be of good qualitySubmitted RNA/DNA samples should be of good quality

• Description of project / sample informationDescription of project / sample information

• It is advisable to have an n of 5-6 for each group to have a good It is advisable to have an n of 5-6 for each group to have a good statistical power for analysisstatistical power for analysis

• If you have only one or 2 samples per group, the core wont be If you have only one or 2 samples per group, the core wont be responsible for data qualityresponsible for data quality

• Turn around time is 2 weeks since the receipt of samplesTurn around time is 2 weeks since the receipt of samples

• You will be notified by email the status of your project and upon You will be notified by email the status of your project and upon completion will send you QC datacompletion will send you QC data

• If a problem arises with the analysis of one or more of your If a problem arises with the analysis of one or more of your specimens, you will be contacted immediatelyspecimens, you will be contacted immediately

Contact InformationContact Information• Mark Gladwin – 301-435-2310Mark Gladwin – 301-435-2310

mgladwin@nhlbi.nih.govmgladwin@nhlbi.nih.gov

• Nalini Raghavachari – 301-435-2304Nalini Raghavachari – 301-435-2304nraghavachari@cc.nih.govnraghavachari@cc.nih.gov

• Tony Cooper – 301-496-8123Tony Cooper – 301-496-8123cooperro@mail.nih.gov

• Xiuli Xu 301-402-2102Xiuli Xu 301-402-2102xux3@nhlbi.nih.govxux3@nhlbi.nih.gov

• Hangxia Qiu 301-451-6253Hangxia Qiu 301-451-6253qiuh2@mail.nih.govqiuh2@mail.nih.gov

• Website Website NHLBI/Vascular Medicine Branch/Genomics CoreNHLBI/Vascular Medicine Branch/Genomics Corecontacts, protocols, status of projects, meetings, tutorials contacts, protocols, status of projects, meetings, tutorials