MyGPCL Login  |  Register    
   Register    Directory     Protocols    Forms     Pricing       
DNA Sequencing with the Roche Genome Sequencer FLX system (formerly 454)
Services | DNA Sequencing Services | Sanger Service Overview | FLX Service Overview | Sanger Sequencing FAQ | Sample Preparation | Methods and Data Delivery | Scheduling and Shipping
 
The Genome Sequencer FLX, based on pyrosequencing technology, provides ultra deep sequencing of 250 bp (average length) segments of DNA, returning approximately 100 Mbases of data per run. The read length, and therefore the total megabases produced, is expected to double by end of summer 2008. Technology and Application overviews are available from 454 Life Sciences.

There are 3 distinct approaches to sample preparation for genetic analysis, each optimal for different project aims.

  1. Shotgun Library preparation is useful for de novo or resequencing of genomic DNA. The DNA sample is randomly fragmented into 200-500 bp pieces. Universal primer adapters are ligated to all fragments. Emulsion micro reactors are created to amplify a single fragment per reactor. Thousands of amplified fragments are sequenced. Analysis software aligns the overlapping reads to determine contigs. The most common DNA input for this process are small genomes, isolated segments of larger genomes or long PCR products (>1 Kb).
  2. Paired End Library preparation provides regions of sequence a known distance apart, allowing for ordering of contigs and analysis of genetic rearrangement. DNA sample is fragmented to 3 Kb segments. Adapters are ligated and the sample circularized. The circularized sample is randomly fragmented and treated as a shotgun library. Fragments with interior circularization adapter sequence represent portions of the sample DNA that are separated by 3Kb. Fragments without adapter sequence are treated as standard shotgun library. Software orders both types of fragments into contigs and uses the paired end information to order contigs into larger scaffolds. The process is valuable to complete de novo assembly or to discover and assess genetic rearrangement.
  3. Amplicon Library preparation provides a means of detecting rare sequence variation. A region of interest, no more than 500 bp, is amplified using specific primers modified to be compatible with the downstream library processing. Amplicons are further amplified in microreactors, a single amplicon per reactor, and sequenced. Software identifies the frequency of variant sequence.

The FLX can also be used to examine gene expression or small RNA profiles by modified library preparation from cDNA samples.

The Bioinformatics Analysis Core has staff trained in appropriate sequence analysis techniques prepared to assist in project analysis. Contact.

FLX projects are complex and require consultation with GPCL staff for study design and project scheduling. Unscheduled drop offs will not be accepted. You must be a registered facility user before submitting any samples for processing. If your laboratory is new to our services, please register here.

Useful information to assist with project planning.