UCI GENOMICS HIGH THROUGHPUT FACILITY Institute for Genomics and Bioinformatics Review October 11,...

UCI GENOMICSHIGH THROUGHPUT FACILITY

Institute for Genomics and BioinformaticsReview

October 11, 2010

UCI GENOMICS

HIGH THROUGHPUT FACILITY

Personnel• Director, Suzanne Sandmeyer, Ph.D.• Manager, Robert Chadwick, Ph.D.• Staff Research Associates

Seung Ah Chung, SRAIII

Valentina Ciobanu, SRAII

Sharmila Mallya, SRAII• Co-Director for Bioinformatics, Pierre Baldi,

Ph.D. • Staff postdoctoral fellow/programmer

Christophe Magnan, Ph.D.

UCI GENOMICS


HT Processing and Affymetrix GeneChip Analysis

Hybridization Oven 640Fluidics Station 450 (3 each)GeneArray Scanner 7GArrayPlex/Beckman FX BioRobot

UCI GENOMICS


Illumina HiSeq Next Gen Sequencer

Illumina GAIIX Next Gen Sequencer

Cbot cluster StationPaired-end Module

MJ Research Tetrad ThermalcyclerAgilent Bioanalyzer 2100Covaris shearer

UCI GENOMICS


Genomics ServicesAffymetrix microarray analysisSample preparation and QCExpression analysisSNP and Copy Number VariationRe-sequencingChIP-Chip

Illumina HT DNA sequence analysisCovaris DNA shearingDe novo & Re-Sequencing of DNARNA-seq (Digital Expression)ChIP-seq (Chromatin sequencing)library preparation (coming soon)

Sample evaluationAgilent Bioanalyzer RNA and DNA analysisNanoDrop RNA & DNA quantificationqPCR of Illumina libraries

WEB REDESIGN

UCI GENOMICS

HIGH THROUGHPUT FACILITYGHTF Analysis PackagesBasic consulting on data analysis approaches (free) & perform defined data analysis routines (recharge basis)

Recharged access to users on 2 facility computers dedicated for data analysis with the following software:•Partek’s Genomic Suite•Affymetrix Expression Consol•Affymetrix Genotyping Console & Integrated Genome Browser•SAS JMP Genomics•Various Web-based & Freeware Application Solutions•Genomics Workbench from CLC Bio

Web based statistical program Cyber-T on Linux Server: http://genomics.biochem.uci.edu/genex/cybert/

Work with the Institute for Genomics and Bioinfomatics: http://www.igb.uci.edu/

http://genomics.biochem.uci.edu/genex/cybert/

http://www.igb.uci.edu/

UCI GENOMICS


Experimental design consultation

Investigator

Institute for Genomics and Bioinformatics

Pierre Baldi/Christophe MagnanRichard LathropPadhraic SmythXiaohui Xie

UCI Statistical CoreRobert NewcombeHal SternRita Petersen

CFCCC Bioinformatics Core

Chad GarnerJenny Wu

Experiment

Analysis

UCI GENOMICS


Fixed expenses:PersonnelTotal recharges $2009-2010: $586,4262010-2011 (est.): $698,085

UCI GENOMICS


Grants:NIH Shared Instrument Grant $500,000 (SBS PI; PB co-PI)

Campus cost sharing:Year 1: $425,000Year 2: $85,000 salariesYear 3-5 $100,000 salaries plus service contract

contribution

Stem Cell Center$360,000 toward HiSeq2000 Annual campus supportCFCCC: $35,000

UCI GENOMICS


UCI GENOMICS


•llumina pipeline software for raw reads, quality scoring, and alignment (ELAND)

•Genomics Workbench can be found here: http://www.clcbio.com/

•MAQ information can be found here: http://maq.sourceforge.net/ •VELVET information can be found here: http://www.ebi.ac.uk/~zerbino/velvet/ •Bowtie information can be found here: http://bowtie-bio.sourceforge.net/tutorial.shtml •A discussion comparing some aligners can be found here:http://massgenomics.wordpress.com/2008/05/14/short-read-aligners-maq-eland-and-others/or http://www.sanger.ac.uk/Users/lh3/NGSalign.shtml

http://bowtie-bio.sourceforge.net/tutorial.shtml

http://massgenomics.wordpress.com/2008/05/14/short-read-aligners-maq-eland-and-others/

http://massgenomics.wordpress.com/2008/05/14/short-read-aligners-maq-eland-and-others/

UCI GENOMICS


Financial Support

Affymetrix Use

CCSG

Charge Backs

InstitutionalSupport

Illumina Use

CC Members

Non-centerMembers

Non-centerMembers

UCI GENOMICS


LeadershipSuzanne Sandmeyer, Ph.D., Biological Chemistry, DirectorRobert Chadwick, Ph.D., Manager GHTFPierre Baldi, Ph.D., Computer Science, Co-Director for Bioinformatics GHTFChad Garner, Ph.D., Director CFC Cancer Center Bioinformatics

Advisory BoardDan Mercola, M.D., Pathology, Director CFCCC Personal GenomicsHung Fan, Ph.D., Director, Cancer Research Institute, Dept. Molecular Biology and

BiochemistryAnthony Long, Ph.D., Dept. Ecology and Evolutionary BiologyPadhraic Smyth, Ph.D., Dept. Computer SciencesBogi Andersen, M.D., Dept. Medicine, Div. of EndocrinologyRobert Newcomb, Ph.D., Dept. Statistics Peter Donovan, Ph.D., Director, Stem Cell CenterLisa Dahm, Ph.D., Director, Clinical InformaticsKyoko Yokomori, Ph.D., Dept. Biological Chemistry

UCI GENOMICS


UCI GENOMICS


Reduced Cluster Density Variability Using cBot vs Cluster Station

UCI GENOMICS

HIGH THROUGHPUT FACILITYImproved Library Quantitation by qPCR Reduces Cluster Generation Variability

UCI GENOMICS

HIGH THROUGHPUT FACILITYGAIIX versus HiSeq 2000 at the Broad Institute

UCI GENOMICS


Accuracy of HiSeq 2000 at the Broad Institute

UCI GENOMICS


NEBNext paired-end genomic library $350 for 10 libraries (primers not included)NuGen Encore paired-end genomic library $680 for 8 librariesEpicentre Nextera paired-end genomic library $625 for 5 librariesIllumina RNA-Seq library $3500 for 8 libraries (Superscript not included)Ambion/NuGen RNA-Seq library $1480 for 8 libraries Epicentre ScriptSeq RNA-Seq library $1200 for 6 libraries

In processNuGen Barcoded RNA-Seq Libraries $3200 for 4 libraries (8 barcodes x 4 RNA)Epicentre Barcoded ScriptSeq Libraries $1400 for 4 libraries (12 barcodes x for Paired End Genomic Libraries take 4 to 6 hours to makeRNA-Seq Libraries take 6 hours (Epicentre) to 3 partial days (Illumina) to make RNA

Library Methods Tested in the GHTF

UCI GENOMICS

HIGH THROUGHPUT FACILITYStandard Curve : Standards from KAPA Library qPCR Kit

Efficiency of the standard qPCR reaction is 98%

UCI GENOMICS


Sample Nanodrop QPCR

ng/ul ng/ul

Nugen DNA lib: SM 65.81 48.77

Nugen DNA lib :VC 60.8 33.66

PhiX control 3.12

RNA seq Illumina 35.51 0.00007

RNA seq 1 Ambion_Nugen Lib kit 9.67 1.2

RNA seq 2 AmbionSecond cycle cDNA_Nugen lib kit 27.22 18.99

Comparison Between Nanodrop and qPCR Quantification

Note that by Nanodrop the Illumina RNA-Seq library gives a good yield but by qPCR the yield was very poor indicating the library is mostly primer artifacts.

UCI GENOMICS


* Whole genome re-sequencing and targeted re-sequencing of genomes of any size and type – from bacteria and viral to humans * De novo assembly of an unlimited number of reads, for genomes up to human size (3 billion bp) * SNP/DIP detection and identification of genomic rearrangements * Digital Gene Expression based on RNA-Seq, including a wide range of downstream gene expression analyses * Discovery of novel transcripts/exons * Ability to work with Expression Arrays and RNA-seq results at the same time, enabling comparison of results * Interactive views of assemblies and derived gene expression data * Small RNA-seq * Tag Profiling, including SAGE screen analyses * Chromatin immunoprecipitation sequencing (ChIP-seq) analysis * Peak finding and peak refinement * Graph and table of background distribution and false discovery rate * Peak table and annotations

Genomics Workbench (CLC Bio)

The Broadcom Distributed Unified Cluster (BDUC) is, as the name suggests, a distributed group of clusters unified by running under a single Sun Grid Engine (SGE) Resource Manager. BDUC consists of 2 subclusters of 40 2core AMD64 Opteron252/2.6GHz nodes running 64bit/CentOS 5. One group of 40 nodes is in the NACS Academic Data Center and another of 40 nodes is in the ICS data center, for a total of 80 nodes / 160 cores. The nodes are interconnected with 1Gb ethernet and have the MPICH and MPICH2 environments for parallel jobs.

BDUC

Sequence Capture Microarrays Can Be Used to Enrich Genomic DNA for Specific Regions of the Genome

Following enrichment and DNA amplification, the enriched DNA regions can be directly sequenced using next-gen sequencers.This can greatly expedite the discovery of causative genes ingenetic linkage association studies.

UCI GENOMICS HIGH THROUGHPUT FACILITY Institute for Genomics and Bioinformatics Review October 11,...

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