Harvard University
“Sanger Sequencing”Sanger DNA Sequencing(Image of ABI)Brand Name underneathApplied Biosystems 3730xl SequencerOur DNA Sequencing division functions both as a small-scale sequencing facility and a center for large-scale sequencing projects with the capacity to analyze over 350,000 DNA samples per year using the best capillary sequencing technology available. The core offers more than 18 years of solid experience in conventional Sanger DNA sequencing, including specialty sequencing and specific supporting services, and reliably provides high-quality services with rapid turnaround and competitive pricing. OverviewOur Sanger sequencing process begins with a cycle sequencing reaction using the Applied Biosystems BigDye v3.1 Cycle Sequencing Kit which employs a fluorescently-labeled dideoxy-nucleotide chain termination method to generate extension products from DNA templates. Extension products are purified using SPRI technology. Subsequently, fragment separation and sequence detection is carried out by capillary electrophoresis on the 96-well capillary matrix of an ABI3730XL DNA Analyzer, followed by post-detection processing. In the final analysis step, a combination of software base calling and manual inspection of the individual trace files is employed to warrant the highest possible quality of the generated data. Upon successful completion of the analysis automated email notifications are sent to our customers who are then able to access their sequencing data securely through our website by logging into our LIMS and downloading the results (chromatograms, text files, and quality files are returned to the customer). (Process Flowchart)Our in-house Laboratory Information Management System (LIMS) tracks each individual sample (or ‘order’) from sample entry and reception through the entire sequencing pipeline, and it allows our customers to submit orders online, check the status of their order(s), and finally to retrieve their data.To ensure consistency in quality during all stages of our process, the core has implemented Standard Operating Procedures (SOPs), stringent multi-level quality controls ( in-process quality controls, routinely performed out-of-process QC exercises, controlled reagent release into production), thorough Failure-Mode-Effect-Analysis (FMEA), daily tracking of quality metrics, and continuous employee training programs for all stages of the process.In the rare event of an equipment failure, of a reagent failure, or of a human error, all affected samples will be automatically repeated at no extra cost for our customers.We provide extensive customer support on an individual basis as well as technical and scientific consultation expertise with the goal of improving the success rate of our customers’ research experiments. Our support includes but is not limited to assistance in template and sample preparation, in the selection of the appropriate sequencing protocol, and in data interpretation and troubleshooting if needed. The core makes sequencing a convenient daily service for the Partners community by providing free local sample-pickup (with the exception of weekends and official MGH Holidays). With our daily courier service between our facility, the MGH main and Charlestown campuses, and the Simches building, samples are delivered within an hour, processed immediately, and data returned within 24 hours (business days only).The sequencing group is capable of processing up to 2500 samples per day. To accelerate larger sequencing projects (typically between 1,000-20,000 samples), operational flexibility combined with rapid changes in production scheduling allows for an even higher daily throughput. Once sequenced, customer samples will be stored for 1 week to enable re-ordering of complete plates or single tubes.=====================================================================================================================Sequencing ServicesIndividual Tube Sequencing This service option accommodates project sizes as small as a single sample. The customer will submit a premix of DNA template (purified plasmids or purified PCR product) and sequencing primer, prepared in a tube as described in our xxx section. Dependent on the template, read lengths of up to 750 bp can be usually achieved.96-well Plate (High-Throughput) Sequencing This service employs the same state-of-the-art sequencing technology as used in our single-tube sequencing Service. Premixed samples (purified plasmids or purified PCR products) are submitted in plate-format (95 samples per plate). Well H12 remains empty to allow for the addition of an in-process control. 96-Well Plasmid DNA Preparation and 96-Well PCR Purification are available as additional plate sequencing services ( link?) to support the submission of plasmid clones (in form of sedimented bacterial cells) or as unpurified PCR products. In these cases, the primer(s) will be submitted separately (in tube- or plate-format) and added to the purified and normalized template(s) by the core personnel. Due to the decreased processing time of samples provided in 96-well format, the core is able to offer a bulk-rate discount. For large-volume orders (> 10 96-well plates), we usually recommend a pilot experiment (1-2 plates typically). Please contact the Core Director for more information on this service.Difficult Template SequencingIn Sanger sequencing, some templates/regions are difficult to sequence when using the standard dye-chemistry techniques and thus often fail to produce satisfactory reads. These templates include GC-rich regions (>60-65% GC content), repeats (di- and tri-nucleotide stretches), hairpin structures (shRNA), long homopolymer stretches (Poly A/T regions), and regions with strong stop sites. While our standard sequencing protocol may perform satisfactorily (but not necessarily reproducibly) for borderline cases, we are recommending our alternative sequencing protocol which can solve many of these problems and increase the read length for a wide range of these types of difficult templates. This service can be added on to both our standard individual tube and our 96-well plate sequencing services for an additional fee. ?Long-Read SequencingBlah blah blahRead lengths up to ….Q20 basesTry to 24-hr turnaround, dependent on volume., 12 samples minimum due to low demand but same equipment time, labor, and reagent cost; different protocol, different operational schedule …Primer Walking & Plasmid ValidationFor DNA sequence analysis of a template/region that is larger than the average read length obtained with our Long-Read Sequencing service, our sequencing group offers sequencing of one or both strands of your insert/PCR amplicon (1X or 2X coverage) or of the entire plasmid. These services include manual design and synthesis of specific primers, editing and assembly of sequence data, comparison to known reference sequence (if available), and manual BLASTn analysis (upon request only). Since service costs can vary due to project specifications as well as specific intrinsic features of the DNA sequence, our Primer Walking and Plasmid Validation projects are handled as custom projects. Please contact xx to request a cost estimate.=====================================================================================Our core facility is equipped with the latest state-of-the-art instrumentation:ABI3730XL 96-capillary DNA Analyzer (3)Tecan Freedom EVO 150 Liquid Handling Robot (1)Eppendorf Mastercycler pro (20)===================================================================================== ................
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