The Mission of the Great Lakes Genomics Center is to support researchers developing and using genomic data for ecology and environmental health. We do this by providing sequencing and gene expression technologies and service for ecology, environment and human health researchers, as well as a support system for analysis and discussion of issues surrounding the use of genomic technologies for environmental health. We also service the larger scientific community with sequencing and transcriptome analysis needs.
Our Vision is to provide cutting-edge technology that will transform our understanding of the Great Lakes and other freshwater ecosystems and provide crucial information for policy makers to best protect human health and the environment.
The PacBio® RS II sequencing system allows scientists to rapidly and cost effectively generate finished genome assemblies, reveal and understand epigenomes, and characterize genomic variation. It achieves the industry’s longest read lengths and highest consensus accuracy.
The PacBio RS II is a Single Molecule, Real-Time (SMRT®) DNA Sequencing System that provides the highest consensus accuracy and longest read lengths of any available sequencing technology. SMRT Sequencing is ideal for de novo assembly,characterization of genetic variation, methylation analysis, microbiology studies, and more.
The instrument features high performance optics, automated liquid handling, and an environmental control center, all directed through an intuitive touchscreen interface. The computational brain responsible for primary data analysis, called the Blade Center, is also included. This allows for seamless integration of performance enhancements through chemistry and software advances.
The GLGC offers library preparations for 16S metagenomics, ChIPseq, small genome resequencing, amplicon sequencing, and small genome RNA sequencing. The MiSeqv3 can generate up to 15 Gb of output with 20-25 M sequencing reads and 2×300 base pair read lengths.
Each MiSeq library prep and sequencing project is highly specific and unique. We strongly suggest consulting with GLGC staff before samples are collected and processed so that the experiment can be set up in the best way possible.
Sample submission requirements are below. All samples submitted for library prep and sequencing must be quantified on the Qubit and quality measures and scores in the table below must be met. Quantification and quality measures can be done by the user or by GLGC for a fee.
Multiplexing is available for most sequencing applications. The number of libraries that can be sequenced on flowcell depends upon the number of samples, coverage needed and genome size.
Library prep and sequencing turn around time is highly variable, depending upon current GLGC work load, sample number, and library type. An estimate of turn around time will be given when samples are submitted.
RNA samples need to be shipped overnight on dry ice and DNA overnight on wet ice.
Our Sanger sequencing services can process plasmid, PCR products, cosmids, phage, and BAC DNA samples. Our 3730 routinely provides up to 800 bases of usable sequence per reaction. We also provide genotyping services for researchers conducting studies with fluorescently labeled primers. We can process microsatellites, AFLPs, and SNPs. All samples must be submitted in either a single tube or 96 well plate format. We offer 2 levels of service: full service and ready to load plates.
Full Service Sequencing
User supplies DNA and primer. We will set up, cycle, clean and analyze the reaction. Each unique DNA template/primer combination is considered to be 1 reaction. BigDye v3.1 is used for all sequencing reactions.
Full Service Microsatellites
User supplies DNA and fluorescently tagged primers. We will set-up, cycle, and analyze the reactions. Reaction set-up information and thermocycler profile needs to be provided by the user. Optimization of reactions must be done by user and is not included in this service. We are calibrated for the DS30 4 color dye set and G5 DS33 5 color dye set and can calibrate for other dye sets if necessary.
Ready to Load Plates – Sequencing and Fragment Analysis: Client performs their own reactions in a 96-well, half-skirted plate, cleans up the reaction, and re-suspends samples in at least 10 /μl of formamide. Wells that do not contain a sample to be sequenced must be filled with 10 /μl of nuclease free water. Please be sure to use plates compatible with an ABI 3730 DNA Analyzer. We recommend these: Dot Scientific, catalog # 951-PCR.
Plates should be delivered to the Genomics Center foil sealed and well labeled. If a sample name other than the well position is needed, provide a corresponding MS Excel spreadsheet organized by columns, ie A1,B1,C1, etc. The plates can only be loaded onto the sequencer in one direction and well position is automatically assigned to the sequence. It is very important to have your MS Excel spreadsheet laid out correctly.
Training is available from GLGC if you would like to set-up your own sequencing reactions and prepare a ready to load plate.
Please contact GLGC 1 day in advance so that we can be ready to load your plates onto the sequencer when they are delivered to the lab.
Things to keep in mind:
The most important factor in DNA sequencing is staring with a clean, pure template. We do not recommend and re-sequencing policies will not apply when sequencing a template with a 260/280 ratio below 1.8 and 260/230 ration below 1.6.
Dilute template in water not TE buffer.
PCR primers need to be removed before the Big Dye Terminator sequencing reaction can be set up. We recommend Qiagen’s QiaQuick PCR clean-up kit. Please also make sure that you only have 1 product from the PCR reaction.
Good PCR primers do not always make good sequencing primers. When sequencing PCR products, you may want to consider 2 different primers for sequencing the region of interest or sequencing from both directions.
You can expect 500-800 bp of high quality sequence when staring with high quality DNA. However, the first 25 – 50 bases are never high quality sequence so primers need to be designed accordingly to be sure you have good sequence over your region of interest.
It is difficult to sequence PCR fragments that are less that 100bp due to limitations of the BigDye v3.1 Terminator chemistry and many PCR column – based clean up techniques. If you need to sequence a small PCR fragment please contact Angie Schmoldt.
The Bioinformatics Team of the Great Lakes Genomics Center is offering various services to researchers and interested parties from both academia and the private sector. The following is a list of services available:
We offer project based collaborations. These can be funded through either a fee-for-services or through joint research grants. We currently are participating in multiple research collaborations through various fields of life sciences also beyond the scope of freshwater sciences like cancer or environmental metagenomics.
We are happy to offer consultation services for data analysis and project design. These can be scheduled either upon request or during our regular office hours. The necessary bioinformatic software tools are either already available by us, or will be implemented as needed.
Bioinformatics support for Next Generation Sequencing (NGS):
NGS services for data analysis are available for various projects like whole genome sequencing (templated or de novo), targeted resequencing, whole transcriptome sequencing (cDNA or RNA based), DNA fragment sequencing derived from Chromosome Immunoprecipitation (ChIP-Seq), DNA Methylation detection (Meth-Seq) and many other potential applications. The Bioinformatics team is available to help investigators with their NGS data analysis or train their staff if needed with off-the-shelf software or newly developed tools.
Access the state-of-the-art computational resources:
Our team has access to bioinformatics computational resources like our own Galaxy Slipstream Server (Link) and priority on our local high-performance computing cluster (Link). Direct access to the computational resources is limited to UWM researchers, but our team is available to use these for external researcher’s data when needed.
We offer bioinformatics training in the form of one-on-one sessions and lectures/seminars. These include a wide range of topics like: introduction to using Linux based computers and biological data types, using basic analysis and visualization tools and complex analysis like those involved in Next Generation Sequencing.
Currently GLGC offers qPCR services utilizing the StepOnePlus qPCR system for gene expression studies at various levels. We will process samples for SYBR green detection only. Primers must be designed and purchased by the user. Prices do not include optimization.
Services include RNA extraction from tissue, cDNA synthesis, and qPCR reaction set-up and SYBR detection. We will accept samples at any step of the process. RNA must have a RIN of 8 or higher and be quantified on the Qubit. Quantification and quality measures can be done by the user or by GLGC for a fee. A qPCR reaction consists of 1 cDNA sample and 1 primer pair or target. All qPCR reactions are ran in duplicate. qPCR reactions are charged by data point which is the average of the duplicate rections.
Ship samples on dry ice, overnight or preserve samples in RNA later.
RNA and DNA Extraction
Currently, services are offered for DNA extraction from water and soil samples and RNA extraction from tissue samples.
Soil and water samples can be stored in sterile collection tubes and stored at -20° until shipped. Tissue samples collected for RNA extractions should be flash froze in liquid nitrogen and stored at -80° until shipped.
Ship DNA samples on wet ice, overnight and RNA tissue samples on dry ice, overnight.
The Great Lakes Genomics Center will quantify and assess quality of DNA and RNA extractions using the Qubit Fluorometer, Agilent Bioanalyzer 2100, and Nanodrop.
The Qubit uses a fluorescence – based method to quantify nucleic acids that is not influenced by contaminants and is therefore one of the most accurate quantification methods available.
The Agilent Bioanalyzer 2100 is the industry standard for RNA quality assessment. A RIN (RNA integrity number) will be provided, based on the 16S/18S ratio.
The Nanodrop 1000 260/230 and 260/280 ratios will provide sample purity information.
RNA samples need to be shipped overnight on dry ice and DNA overnight on wet ice.
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