Next-generation sequencing is a powerful means to profile genome methylation status at single-nucleotide resolution. Historically, this has been accomplished using sodium bisulfite. Exposure of DNA to sodium bisulfite converts cytosine (C) – but not methylated cytosine (C*) – to uracil (U). Subsequent amplification of the DNA by PCR converts U to T.
More recently, a two-step enzymatic process has been developed that achieves the same endpoint but avoids much of the DNA damage associated with sodium bisulfite conversion. Once such conversion has been accomplished by either method, the original methylation state – C vs C* – can be read out as the presence of a T vs C at a given CpG.
DNA Methylation Options
Whole Genome Bisulfite Sequencing (WGBS): WGBS combines sodium bisulfite conversion with high-throughput DNA sequencing to determine DNA methylation states at individual cytosines. Using this technique, unmethylated cytosines are converted into uracils and then into thymines after PCR.
Reduced Representation Bisulfite Sequencing (RRBS): RRBS is a bisulfite-based protocol that reduces the amount of sequencing required by focusing on a subset of the genome where the majority of the DNA methylation occurs.
Enzymatic Methyl-seq (EM-seq): The enzymatic methyl-seq workflow developed at NEB provides an alternative to bisulfite sequencing. The NEBNext Enzymatic Methyl-seq Kit (EM-seq) combines NEBNext Ultra II reagents with two enzymatic steps to construct Illumina libraries that accurately represent 5mC and 5hmC within the genome. EM-seq libraries result in minimal DNA fragmentation and biases compared to WGBS.
Targeted EM-seq: Targeted EM-seq starts with the same two-step enzymatic conversion to generate indexed libraries in which the unmethylated cytosines have been converted to thymines. These converted libraries are then hybridized to a probe panel comprising all iterations of the methylome for your region of interest. This enzymatic conversion, coupled with probe capture, yields high-quality methylome data with relatively low sequencing cost.
Start a Project
Contact us a next-gen@umn.edu for project support or questions. If your project has unique requirements, higher complexity, or needs optimization, our Custom Services can do non-routine protocols not offered through our Standard Services.
Please inquire about library creation rates at next-gen@umn.edu.
Submission
How to Order
1. Please contact next-gen@umn.edu for project specifications.
2. Once project details are finalized, complete the appropriate submission form for submitting samples or submitting client-made libraries and email to next-gen@umn.edu.
Illumina Sequencing Request Form (Libraries)
Illumina Sequencing Request Form (Samples)
Shipping Instructions
Samples should be frozen and shipped on dry ice in a 96-well plate. We recommend using plate tape to seal the wells. Place the plate inside of a plastic bag prior to placing on dry ice. Please give advance notice of submission date and time so staff can be prepared to receive samples. If shipping samples from outside the University of Minnesota, ship via express shipping carrier on dry ice to the address below.
Samples can be dropped off at one of our campus locations
1-210 Cancer & Cardiovascular Research Building (Minneapolis campus)
20 Snyder Hall (St. Paul campus)
Samples can be shipped to the following address:
Please send the tracking information to next-gen@umn.edu.
UMN Genomics Center
ATTN: Elyse Froehling
3510 Hopkins Place N.
Building 4 Suite W402
Oakdale, MN 55128
612-625-7736
Deliverables
Data Release
There are four options for transferring data from the UMGC to clients: 1) delivery to the Minnesota Supercomputing Institute’s (MSI) high-performance file system, 2) download from a secure website, 3) download with Globus, or 4) shipment on an external hard drive. Please indicate your data delivery preference when placing an order for sequencing.
1. MSI storage
Internal clients have their data released to MSI's Shared User Resource Facility Storage (SURFS). Delivered data will be located in the "data_delivery" folder in your group's folder on MSI's primary filesystem (home/GROUP/data_delivery/umgc). MSI does not charge for SURFS storage costs, but files expire and are removed one year after they've been delivered. Files should be copied to other MSI storage locations such as Tier2, Tier3, or your group's "shared" folder before they expire.
2. Web download
Internal clients that opt-out of MSI storage and external clients can download their data from a secure website using either a web browser or a command-line download tool, complete instructions are provided in an email from the UMGC. The client’s data is available for download for 30 days, after which the data will be removed from the data download website and the client takes responsibility for storing the data.
3. Globus
Internal and External clients can use Globus to download their data. This is the recommended method for external clients to download large datasets.
4. Hard drive
External clients may have data shipped on a hard drive purchased by the UMGC and invoiced to the client at a cost of $250 per hard drive.
Data Recovery
The UMGC archives most customer data for a year and some datasets are retained for 5 years or more. If you need a dataset re-delivered email a request to next-gen@umn.edu to initiate data recovery. The UMGC does not provide any guarantee that data can be successfully recovered from the archive.