The Center for Genome Innovation is pleased to announce the addition of Sanger Sequencing and STR Analysis (new service to support NIH sample Authentication requirements) as part of our service portfolio effective Monday, November 28th, 2016.
Samples for Sanger Sequencing and STR Fragment analysis can be submitted directly to the CGI (Beach Hall, room 201), or by using the DropBox service (see below). All submissions must be accompanied by completed online form submission found here
See table below for a complete list of service types as well as descriptions for each service.
Sanger Sequencing DropBox Locations and Submission Policies
Small black refrigerators will serve as DropBox locations in both the Biology/Physics building (G05) and Beach Hall (CGI room 209; outside of the main lab, room 201). Samples will be collected daily from both locations at 1:00pm by CGI staff for processing that day. Samples must be placed inside a freezer box or in a zip top plastic bag CLEARLY labeled with YOUR NAME, PI name and submission date. NOTE: Samples may be picked up later than 1:00pm daily, but never earlier than 1:00pm.
- Be sure to complete the online submission form before leaving your samples in these fridges!
- Collection logs are found on the top of each fridge to document daily pickup.
- All samples being dropped of must comply with CGI submission guidelines (see below)
- Customers submitting samples for Run-Only (no CGI Labor) sequencing must inform the CGI at least 24 hours prior to sample submission so that similar project submissions can be coordinated with the run (to expedite data delivery).
- High volume sample submission (i.e. >48 sequencing reactions) should be brought to the CGI’s attention at least 24 hours prior to sample submission in order to ensure timely processing.
Sanger Re-sequencing Policy
There are many different submission types for Sanger sequencing, ranging from “Run Only (no Labor)” to full service sample preparation and sequencing. Please be advised that no-cost re-sequencing for failed sequencing runs or fragment analysis runs will only be allowed for DNA samples that can be validated for concentration and integrity by the CGI prior to start of project. Customer premixed template and primer samples for Sanger can not be internally verified for concentration and integrity therefore any sequencing failures that are not due to instrument error will be re-sequenced at customers expense.
Sample PreMix Guidelines
Sanger Sequencing Reactions follow the BigDye Terminator v3.1 protocol for 10uL reactions. Customers that submit premixed primer, template [and water] MUST prepare all premix reactions in individual PCR tubes or 8-strip PCR tubes (preferred) according to the following recipe (makes 7.75uL of total volume):
- DNA Template: Up to 6.75uL volume of template meeting the specifications for total ng input. See Table in Sample Submission Guidelines section for input specifications based on template length
- Sequencing Primer: 1uL of a 3.2uM sequencing primer
- Water: [if necessary] bring up volume to 7.75uL with Nuclease Free Water
Commonly Used Sequencing Primers
The CGI will provide commonly used sequencing primers at the customer’s request for full service sample submission reactions. Here are the primers and their sequences currently available:
M13F: 5’ – GTAAAACGACGGCCAG – 3’
M13R: 5’ – CAGGAAACAGCTATGAC – 3’
T7: 5’ – GTAATACGACTCACTATAGGGC – 3’
SP6: 5’ – TTTAGGTGACACTATAGAATAC – 3’
T3: 5’ – AATTAACCCTCACTAAAGG – 3’
Sample Submission Guidelines for Template Input
- Customers preparing premixed template and primer samples must follow the input recommendations for total template amount set forth in this table:
- Full service sample processing includes template quantitation by the CGI using Qubit. Concentration measurements reported by the CGI using this method will supersede customer NanoDrop measurements. If necessary, customer may be contacted to submit more template if not enough is supplied, which may delay data delivery.
Plasmids submitted for Sanger sequencing MUST be quality checked by the customer for the presence of insert and/or plasmid purity by restriction enzyme digestion. The CGI can assess plasmid integrity by Genomic DNA Agilent TapeStation analysis for an additional cost. The CGI is not responsible for sequencing failures that can be attributed to poor template quality.
Short Tandem Repeat (STR) Fragment Analysis
Effective January 2016, the National Institutes of Health (NIH) Office of Extramural Research (OER) revised grant application requirements as part of the program to “Enhance Rigor and Reproducibility.” Included in this requirement are four components the NIH has considered important for enhancing rigor and transparency. Among these is the “Authentication of Key Biological and/or Chemical Resources”, which must be outlined in all new grant applications and implemented in grants funded from 2016 onward. To summarize the full description1, the NIH requires that resources used to conduct NIH funded research must be authenticated on a regular basis. These resources include all cell lines and other biologics, including but not limited to primary cell lines, established cell lines and those obtained from another source regardless of the source (company with their own sample authentication2, non-NIH funded material source, etc). Specifically, “If key resources have been purchased or obtained from an outside source that provided data on prior authentication, the investigator is still expected to provide their own authentication plans for these key resources.” Moreover, the authentication requirement applies to cell lines and biologics that may differ from laboratory to laboratory or may differ over time (e.g. in culture).
The NIH has not set a standard for this authentication; rather, the scientific community is empowered to provide this standard. However, the NIH has provided a recommendation that cell lines may be authenticated by chromosomal analysis and/or short tandem repeat (STR) profiling.
The CGI and Chromosome Core (Institute for Systems Genomics) provide several services to meet this new NIH requirement. Our newest and most affordable service is forensic-standard STR typing of samples. Within our portfolio, we also offer more detailed assessment of samples, including manual karyotyping and Affymetrix SNP Cytoscan Arrays.
1 The quality of the resources used to conduct research is critical to the ability to reproduce the results. NIH expects that key biological and/or chemical resources will be regularly authenticated to ensure their identity and validity for use in the proposed studies. Key biological and/or chemical resources may or may not be generated with NIH funds and: 1) may differ from laboratory to laboratory or over time; 2) may have qualities and/or qualifications that could influence the research data; and 3) are integral to the proposed research. These include, but are not limited to, cell lines, specialty chemicals, antibodies and other biologics. Researchers should transparently report on what they have done to authenticate key resources, so that consensus can emerge.
2 http://grants.nih.gov/reproducibility/faqs.htm#4850: “If key resources have been purchased or obtained from an outside source that provided data on prior authentication, the investigator is still expected to provide their own authentication plans for these key resources.”