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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).
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]
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 ...
Newbler can run via a Java GUI (gsAssembler) or the command line (runAssembly). It works natively with the .SFF data output by the sequencer, but is also able to accept FASTA files, containing nucleotide sequences, with or without quality information, and FASTQ files.
Created Date: 8/30/2012 4:52:52 PM
HTSeq [10] The Python script htseq-qa takes a file with sequencing reads (either raw or aligned reads) and produces a PDF file with useful plots to assess the technical quality of a run. mRIN [11] - Assessing mRNA integrity directly from RNA-Seq data.