genomics core facility at unh: high-throughput sequencing on the illumina hiseq 2500 platform...
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Genomics Core Facility at UNH: High-Throughput Sequencing on the Illumina HiSeq 2500 Platform
Project Consultation
Sample Submission
Library Creation
Illumina Sequencing
Data Delivery
Analysis/Bioinformatics
Overall Process:The Hubbard Center for Genome Studies (HCGS) leads the development of comparative and environmental genomics research, with a special emphases on novel model species. The Genomics Core Facility (GCF) at HCGS provides services in high-throughput DNA sequencing and a wide variety of technical assistance and support to our international user base. The HCGS and GCF are integrated with the Bioinformatics Core Facility to support these activities. The GCF plays a leading role in education and training in genomics through seminars, short courses, lab rotations, sabbaticals, training, and distribution of protocols.
About us:
Krystalynne Morris, Stephen D. Simpson and W. Kelley Thomas, University of New Hampshire
Project Consultation:During the project consultation phase, you will meet with the HCGS Genomics Core Facility team. We will talk about the overarching research questions of the project and the experimental design. We will talk about the samples, number of replicates, coverage needs, and strategies to maximize amount of data. We will conclude with an understanding of the expectations, need for downstream analysis, and any specialized bioinformatics needs from the Bioinformatics Core Facility
Instrumentation:
cBot
HiSeq 2500
Sample Submission:Samples submitted to the GCF include DNA, RNA, and amplicons. For the best results, the DNA and RNA should be of high quality and show little or sign of degradation. DNA and RNA sample should be suspended in water or buffer (TE or Elution), and free of inhibitors.
Low Quality DNA
Low and High Quality RNA
Library Creation:The type of libraries generated will depend on the research question. The most frequently requested libraries are TruSeq DNA, TruSeq Stranded RNA, Nextera DNA, amplicon, and Genotyping by sequencing.
Lane 1 – LadderLane 2 – LibraryLane 3 – BlankLane 4 – gDNA (note the HMW Band)Lane 5 – Post Covaris shearingLane 6 – Post Adapter LigationLane 7 – Post PCR Ampure discarded wash
nM = 2.491
Most libraries can be quantified using digital PCR, which allows to more precisely and accurately cluster the libraries on the flow cell.
Poor/Good quality RNA
Library QC on the Bioanalyzer
Illumina Sequencing:Once the libraries pass QC and the concentration is determined, they are ready for Illumina sequencing. The cBOT chemistry and process anneals the library to the flow cell, and depending on the chemistry for the run, clusters will be generated within the flowcell while on the cBot or on the HiSeq 2500.
Data Delivery:When the run has finished, the raw output is converted to the Fastq format and deposited on our server. New users will receive two emails log in information for the server (one for the user name, and another for the password). Then you will receive a link to your data for download.
Analysis & Bioinformatics:
The Bioinformatics Core Facility is happy to assist with downstream analysis once the data is generated. Please visit their poster to learn more!
Contact us: [email protected], [email protected], [email protected]