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Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequencing relies on light detection based on a chain reaction when pyrophosphate is released. Hence ...
Standard flowgram format (SFF) is a binary file format used to encode results of pyrosequencing from the 454 Life Sciences platform for high-throughput sequencing.SFF files can be viewed, edited and converted with DNA Baser SFF Workbench (graphic tool), or converted to FASTQ format with sff2fastq or seq_crumbs.
The pyrosequencing method is based on the detection of the pyrophosphate release on nucleotide incorporation. Before performing pyrosequencing, the DNA strand to sequence has to be amplified by PCR. Then the order in which the nucleotides have to be added in the sequencer is chosen (i.e. G-A-T-C).
The principle of Pyrosequencing was first described in 1993 [1] by combining a solid support with an engineered DNA polymerase lacking 3´to 5´exonuclease activity (proof-reading) and luminescence real-time detection using the firefly luciferase. All the key concepts of sequencing by synthesis were introduced, including (1) amplification of ...
Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence reads. [81] This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.
Created Date: 8/30/2012 4:52:52 PM
Download as PDF; Printable version; In other projects ... It is designed specifically for assembling sequence data generated by the 454 GS-series of pyrosequencing ...
mothur is an open source software package for bioinformatics data processing. [7] The package is frequently used in the analysis of DNA from uncultured microbes. mothur is capable of processing data generated from several DNA sequencing methods including 454 pyrosequencing, Illumina HiSeq and MiSeq, Sanger, PacBio, and IonTorrent. [8]