Facilities & Resources

UR Genomics Research Center
The UR Genomics Research Center (URGRC) located at the James P. Wilmot Cancer Center provides core laboratory support, technical advice, assistance with experimental design and data analysis for investigators using high-throughput genomic sequencing, genotyping and gene expression in their research programs. The URGRC currently provides support for RNA-Seq, CHiP-Seq, Exome/Genome sequencing,Single-cell genomics applications, qRT-PCR, Affymetrix microarrays, processing of RNA/DNA, as well as custom applications and approaches. In addition, URGRC staff lead collaborative projects with URMC investigators to develop new methodologies and incorporate emerging genomic technologies into the URGRC workflow. The URGRC has a faculty-level director, a faculty-level Associate Director, a Lab Manager, a Technical Associate, four technicians and two Bioinformaticians/data analysts. Dedicated computational support for hardware, data analysis and storage of high-throughput sequence data is provided by the Center for Integrated Research Computing (CIRC).

 

The following major equipment is located in the URGRC: one Illumina HiSeq2500 high-throughput DNA sequencer, one Illumina MiSeq DNA sequencer, one Fluidigm C1 Single-Cell AutoPrep System, one QuantStudio 12K FLEX real-time PCR instrument with autoloader, microfluidic card module, and open array module for digital PCR, one Sage Science PippinPrep size fractionation system, two ABI 9700 PCR machines, three BioRad DNA Engine PCR machines, CAS 1200 liquid handling robot, two Agilent 2100 Bioanalyzers, one Agilent TapeStation, one BioRad gel documentation system, one Qubit Flourometer, and multiple NanoDrop ND-1000 spectrophotometers.

 

Methods

DNASE Treatment

Residual genomic DNA was removed from purified RNA with TURBO DNASE (Life Technologies, Grand Island, NY). Briefly, total RNA was diluted in 1x TURBO DNase buffer along with 1ul of the TURBO DNase enzyme in a final volume of 50uL. The reaction was incubated at 37C for 30 minutes followed by DNase inactivation with addition of 0.1 volume of DNase inactivation reagent. The inactivation was carried out for 5 minutes at room temperature (22-26C). Total RNA quantity was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality was assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA).

 

mRNA-seq

Total RNA was isolated using the RNeasy Plus Kit (Qiagen, Valencia, CA) per manufacturer’s recommendations. RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer’s protocols. Briefly, mRNA was purified from 100ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3` adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100nt are generated for each sample and aligned to the organism specific reference genome.

 

Stranded mRNA-seq

Total RNA was isolated using the RNeasy Plus Kit (Qiagen, Valencia, CA) per manufacturer’s recommendations. RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality was assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). Illumina compatible library construction was performed using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) per manufacturer’s protocols. Briefly, mRNA was purified from 100ng total RNA with oligo-dT magnetic beads and chemically fragmented. First-strand cDNA synthesis was performed using random hexamer priming followed by second-strand cDNA synthesis using dUTP. End repair and 3` adenylation was performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100nt were generated for each sample and aligned to the organism specific reference genome.

 

Low Input RNA-seq

Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA) or Arcturus Pico Pure kit (Life Technnologies, Carlsbad, CA) per manufacturer’s recommendations. RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). 1ng of total RNA was preamplified with the SMARTer Ultra Low Input kit v2 (Clontech, Mountain View, CA) per manufacturer’s recommendations. The quantity and quality of the subsequent cDNA was determined using the Qubit Flourometer (Life Technnologies, Carlsbad, CA) and the Agilent 2200 TapeStation. 5ng of cDNA was used to generate Illumina compatible sequencing libraries with the NexteraXT library preparation kit (Illumina, San Diego, CA) per manufacturer’s protocols. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100nt were generated for each sample and aligned to the organism specific reference genome.

 

Whole Exome Sequencing

Purified genomic DNA was quantified using the Qubit Flourometer (ThermoFisher, Waltham, MA) and quality was assessed with the gDNA tapestation assay (Agilent Technologies, Santa Clara, CA). Whole exome libraries were generated using SureSelectXT Whole Exome v5 (+UTRs) per manufacturer’s recommendations (Agilent Technologies, Santa Clara, CA). Briefly, 4ug of good quality genomic DNA was sheared with Covaris S2 (Covaris, Woburn, MA) to a average peak size of 250bp followed by end repair, poly adenylation, Illumina adaptor ligation, and purification with AmpureXP (Beckman Coulter, Brea, CA). Library amplification was carried out with 250ng of purified DNA with 4 cycles of PCR followed by AmpureXP purification (Beckman Coulter, Brea, CA). Exome probe hybridization capture was performed per manufacturer’s protocols. Exome target enriched samples were quantified using the Qubit Flourometer (ThermoFisher, Waltham, MA) and library sizing was assessed with the high sensitivity Bioanalyzer 2100 DNA assay (Agilent Technologies, Santa Clara, CA). Whole exome libraries were normalized, pooled and 2x100 pair end sequencing was performed with the HiSeq2500 (Illumina, San Diego, CA). Raw read data generated from the HiSeq2500 sequencer were demultiplexed using configurebcl2fastq.pl version 1.8.4. Quality filtering and adapter removal was performed using Trimmomatic version 0.32 with the following parameters: "SLIDINGWINDOW:4:20 TRAILING:13 LEADING:13 ILLUMINACLIP:adapters.fasta:2:30:10 MINLEN:25". Processed/cleaned reads are then aligned to the human genome reference hg19 with SHRiMP version 2.2.3. Variant analysis was performed using SAMtools.

 

NGS Data Processing and Alignment

Raw reads generated from the Illumina HiSeq2500 sequencer were demultiplexed using configurebcl2fastq.pl version 1.8.4. Quality filtering and adapter removal was performed using Trimmomatic version 0.32 with the following parameters: "SLIDINGWINDOW:4:20 TRAILING:13 LEADING:13 ILLUMINACLIP:adapters.fasta:2:30:10 MINLEN:15". Processed/cleaned reads are then mapped to the UCSC XXX genome build with SHRiMP version 2.2.3 with the following parameters: "--qv-offset 33 --all-contigs". Differential expression analysis is performed using Cufflinks version 2.0.2; specifically, cuffdiff2 and usage of the general transfer format (GTF) annotation file for the given reference genome with the following parameters: "--FDR 0.05 -u -b GENOME".

 


Please contact the URGRC with any questions you have have.