454 Sequencing
Overview of the Genome Sequencer FLX Titanium Sequencing System
The Genome Sequencer (GS) FLX System by 454/Roche (my454.com) is a DNA sequencing system involving preparing, amplifying, and sequencing of a library of DNA fragments in a massively parallel fashion. The GS FLX System workflow can be divided into five general steps, from the input of sample to the analyzed output: Library Preparation, emPCR Library Amplification, Sequencing, Data Processing, and Data Analysis.
The five general steps can briefly be described as follows:
- Library Preparation: The first step of the process is to prepare a DNA library from the sample input. The DNA library must be composed of fragments appropriately modified for amplification and sequencing in the Genome Sequencer FLX System. Several library preparation methods are offered, depending on sample type and throughput required. All include the modification of each DNA fragment by adding special sequences (adaptors) that will be recognized in later workflow steps.
- emPCRLibrary Amplification: Once the library is constructed, the DNA fragments are immobilized onto beads. Beads are amplified and enriched, resulting in the majority of the beads carrying a single DNA fragment. Each bead is isolated in the aqueous phase of a water in oil micelle (emulsification), along with the amplification reagents, for the clonal amplification of the DNA fragments. Amplification is carried out in bulk, resulting in millions of individual beads that are each coated with millions of clonal copies of different amplified DNA fragments.
- Sequencing: After amplification of the library, the DNA-carrying beads are loaded into the wells of a Pico Titer Plate (PTP) device such that each well contains only one DNA bead. The loaded PTP device is then inserted into the Genome Sequencer FLX Instrument and sequencing reagents, including each nucleotide, are sequentially flowed over the plate in cycles. Each cycle generates light that is converted into digital images captured by the camera.
- Data Processing: These digital images, or raw data, are processed by the GS Run Processor. This software application encompasses all the steps required to convert raw data into basecalls and quality scores. The processing results in FASTA and Standard Flowgram Format (SFF) files suitable for use in downstream analysis applications and upload to public DNA sequence databases.
- Data Analysis: Depending on the sample type and the experimental design, several data analysis software have been tailored to generate the final output. These are the GS De Novo Assembler for de novo sequencing and assembly, the GS Reference Mapper for Resequencing and NimbleGen Sequence Capture, GS Amplicon Variant Analyzer (AVA) for Amplicons. FASTA raw reads can be used for Metagenomics and 16S sequences.
We offer services on all these parts for the purpose of de novo genome sequencing, transcriptome (cDNA) and targeted sequencing (amplicon/PCR products).
Page manager: Inger Ekström
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Publisher: Department of Biology
Last modified 18 Oct 2011
Tomas Johansson
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MEMEG
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Björn Canbäck
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Eva Friman
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MEMEG
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