Roche FLX (454)
The IGSP Sequencing Facility is pleased to offer services on our Roche GS-FLX, aka 454 since March, 2008.
454 Sequencing is excellent for producing relatively long reads (100 bp, 240 bp, 385 bp) in large numbers (100-400 Mbp). This technology is used for elucidation of the following types of sequence:
De Novo Sequencing, Amplicon Sequencing, Resequencing, Transcriptome Analysis, Gene Regulation Studies, Metagenomics and Microbial Diversity, and Paleogenomics.
Steps to Running your 454 Project at the Duke IGSP Sequencing Facility
1. Decide what type of project you would like to do. The 454 Website has a good bit of applications type advice.
2. Review our current 454 Price List.
3. Contact Lisa Bukovnik to discuss your project.
4. Prepare your sample according to 454 DNA Prep for gDNA and cDNA samples. The lab does not offer cDNA synthesis as a service. DO NOT RELY ON NANODROP TO GIVE AN ACCURATE MEASURE OF dsDNA CONTENT. Molecular methods such as picogreen are much more precise. If you desire amplicon sequencing look at the Amplicon Application Note on the 454 website. Sample prep. is quite different for amplicons, and requires a bit more up-front work for the customer, but there is no library prep. charge in the Facility.
5. Submit your sample to the Facility to get in the queue. If you are mailing a sample to us, send it well-padded and on dry ice. Each sample is handled with custom care, so turn-around time varies.
6. 454 data are relatively easy to work with. You will receive 3 files for each sample: reads (.fna), quality scores (.qual), and standard file formats (.sff's). You can use the 454 Newbler Assembler and Mapper programs to assemble and align your sequences. For Duke customers, access to this software is included free of charge.
7. The customer should immediately back up her/his data upon receipt. Image files will stay available for 10 business days if the customer wants images backed up, and the customer should provide an external hard drive for doing so. Data management beyond distribution of reads will be charged $30/hour. Unfortunately, the Facility is not yet staffed for bio-informatics support.