Georgia Genomics Facility

• General Information
  • Home
  • Facility Description
  • Contacts
  • Publications
  • Current and Previous Projects
• Capillary DNA Sequencing & Genotyping
  • Overview
  • Sample Prep
  • Costs
  • Submit Samples
• 454 DNA Sequencing
  • Overview
  • Specific Information
  • Sample Prep
  • Multiple Samples, Run Expectations & Guarantees
  • Costs
  • GS FLX 454 flyer (pdf)
• Genotyping
  • BeadXpress
  • LightCycler / Real-Time PCR
  • TaqMan OpenArray Genotyping System
• Other Equipment Available
  • Agilent 2100 Bioanalyser
  • epMotion Automated Pipetting
  • MultiNA
• Education, Seminars, and Outreach
  • Upcoming Seminars

Specific Information

There are 4 major phases to 454 sequencing:  a) library preparation, b) emulsion PCR (emPCR), c) sequencing, and d) data analysis.  Users can submit individual DNA samples prior to any of these steps, after step a, or after step b.  In general, sequencing costs are about 50% for reagents, and 50% for personnel time, machine time, etc.  Although researchers are welcome to do their own library preps or even emPCR, they will be assuming significant risk in the final outcome of the data and very few researchers will find it economical to take on these aspects/risks.

The GGF will make detailed protocols available to academic users who request them.  We have made significant changes to the Roche library prep protocols and we will submit these for publication in the near future.  Our library prep protocols work better, faster, and cheaper than the Roche protocols.  We will hold training seminars and potentially some workshops to train users in these protocols, but the strides in efficiency we have made in library preparations allow us to create libraries at costs that few users will be able to achieve.  The main reason users might want to make their own libraries is to do very large numbers of samples (>100’s) or to do something beyond what we can support (i.e., a very weird one-time protocol).

emPCR requires some specialized equipment, expensive reagents and a significant investment in training, as well as time for each sample.  For example, the reagents used in emPCR cost ~$1500 for a full-run.  The GGF will make detailed protocols available to academic users who request it, but we only recommend serious power-users to undertake this path.  We have tried modifying the Roche protocols and have generally failed at that; so we recommend sticking to the known protocols.  We will give one-on-one lessons for users who choose this path.

All data are delivered via secure ftp (sftp).  Users are assigned password protected folders.  All users within a PI’s lab usually share a single folder, but independent folders can be obtained if requested.

Fasta and quality files will be posted to the sftp and can be copied to DVD upon request.  Data will be trimmed and sorted by MID-tag groups (if MID-tags are used). Additional analyses require additional time, and thus incur additional costs.  At this point, GGF staff can provide only limited bioinformatics services.  However, all Roche/454 software (i.e., Newbler [for de novo assembly], Amplicon Variant Analyzer, and Reference Mapper) are freely available to GGF users.  We will gladly provide this very powerful software, but can offer only limited free support for software installation & use.  We are also coordinating with UGA faculty, the Institute of BioInformatics (IOB), and the Research Computing Center (RCC) at UGA to provide access to a wide variety of bioinformatics tools and resources.  If researchers need bioinformatics support, they should contact the GGF as early in the project planning phase as possible so we can provide guidance about resources required and other possible collaborators who may provide the bioinformatics support needed.