Sequencing: Sanger, Next-generation, and Protein
Sanger sequencing: has been the method of choice for DNA sequencing since the mid 1980’s. The UMGC has been providing this service since its inception in 2000 using Applied Biosystems (ABI) instrumentation and reagents. The method is useful for targeted sequencing of DNA from virtually any source. The method uses specifically designed sequencing primers to amplify a template of interest using the Sanger di-deoxy terminator method. As a terminator base is incorporated into the growing sequence, it terminates the growing DNA molecule and has a unique fluorescent tag that corresponds to one of the four nucleotides. The fluorescently tagged DNA fragments are electrophoresed to assign position in the sequence using ABI’s 3730xl sequencer. The 3730xl is a 96-well capillary electrophoresis instrument. Turnaround time for DNA sequencing results is 72 hours. Traditional Sanger sequencing is a ubiquitous technology that is useful for mutation analysis, plasmid sequencing, BAC sequencing, amplicon analysis and targeted genomic DNA sequencing.
Next-generation sequencing: The UMGC maintains 2 platforms for Next-generation sequencing applications—the Illumina HiSeq 2000 and Illumina MiSeq. Next-generation sequencing protocols are rapidly becoming the method of choice in many areas of genomic analysis. Next-generation sequencing has applicability in many areas of genomic research including: Whole genome sequencing, de novo sequencing, SNP Genotyping and CNV analysis, exome sequencing, small RNA-seq, epigenetic analysis (Methyl-Seq, ChIP-Seq), targeted re-sequencing and RNA-Seq. Both instruments leverage Illumina’s proprietary Sequencing-by-Synthesis (SBS) chemistry that features massively parallel short read (50-150 bp) outputs. The current configuration of the instrument allows both single read (appropriate for basic RNA-seq expression experiments) and paired end reads (appropriate for RNA-seq where splice variant/transcript isoform data is desired). The HiSeq 2000s and MiSeqs together enable a diverse and broad array of applications including whole genome sequencing, trancriptomics and epigenomics.
Protein sequencing: The UMGC uses an ABI Procise sequencer to perform N-terminal protein sequencing for various types of samples. We offer sequencing for HPLC fractions, electro-blotted gel fractions or isolated proteins in solution. In addition, we offer PVDF adsorption using ABI Prosorb technology. These solutions give high repetitive yields and effective sample sequencing at competitive rates.
Next-generation workflows for each system are similar but unique. A summary is below.
1. Consult with UMGC staff to outline project goals and decide which type of sequencing platform to use.
2. Nucleic acid (extracted by the researcher or possibly by UMGC) is submitted to the UMGC for QC.
3. The UMGC will perform sample QC using NanoDrop, Agilent Bioanalyzer, PicoGreen assay, or RiboGreen assay. At this point, the client decides whether or not to proceed with the project.
4.The UMGC will perform library creation and cBOT cluster generation.
5. Sequencing run is performed on specified platform with primary QC analysis.
6. Data assembly performed by Minnesota Supercomputing Institute staff with final release of data file.
1. Client isolates and cleans up PCR products, plasmids, BACs or genomic DNA using Qiagen or Millipore kits.
2. University of Minnesota researcher creates an account in UMGC’s online order system and completes the online order form completely prior to submission. Non-University of Minnesota researchers can request a submission form.
3. Client selects sequencing option and submits form(s) and samples to UMGC.
4. UMGC staff performs sequencing reactions and runs samples on the ABI 3730xl DNA analyzer.
5. UMGC staff reviews data files and provides data to researchers.
Next-generation Includes QC, library preparation, and sequencing. Costs for next-generation sequencing projects are heavily dependent on platform and depth of read required.
DNA Sanger sequencing reactions are priced based on the type of template to be sequenced and the processing option selected.