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 Analysis Principle - Illumina Genome Analyzer
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Illumina's Genome Analyzer attaches randomly fragmented genomic DNA on a flow cell, then reads 75bp sequences at the end of DNA fragments.
DNA fragments are distributed at high density on a flow cell, and 100 million data can be generated at one run.
< 75bp  approx. 100 million = approx. 7.5 billion bp >
* With paired-end method, the both end can be read, so the amount of the generated data will be double.
Analysis is performed using a flow cell which consists of 8 lanes. Orders can be available from one lane according to the required data volume.
Guaranteed volume per one lane is 300Mbp for single end method
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Single Read Analysis
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 Sample Preparation
Provided sample DNA will be fragmented into approx. 200bp size and two kinds of adaptors will be attached to both end.
DNA fragments will be bound and amplified on the flow cell using adaptor sequences and form more than 70 million clusters (assemble of the same DNA fragments).
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Sequencing Analysis
After adding sequencing primers, paratactic large quantity sequencing will be performed on a flow cell by repetitive steps of base extension and fluorescence readout.
<1Cycle>
- Sequencing reagent addition
- One base extension reaction
- Non-reacted base elimination
- Capture of fluorescence signal readout
- Elimination of protecting group and fluorescence
Full length will be analyzed by repetitive cycle reactions.
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Paired End Analysis
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Two kinds of analysis, single-end and paired-end, are available for the analysis by Genome Analyzer.
The sequence at one end of DNA fragment will be read in single-end analysis, while sequencing will be performed for
both end in pair-end analysis. By pair-end analysis, double volume of data will be obtained than single-end analysis and the data of approx.
200 - 300 bp fragments' both end can be obtained as linked information, so suitable for assemble and mapping.
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Analysis Principle - Roche Genome Sequencer FLX
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Sequencing by Genome Sequencer FLX can determine approx. one million data (up to 400bp) per one read, i.e. approx. 400Mbp analysis can be available at one run.
Analysis per divided gasket region may be available depend on your analysis data volume.
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Single Read Analysis
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Sample Preparation
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Genome DNA or BAC will be firstly fragmented to the size between 300 and 800 bp, then two kinds of short adapters - specific for 3' and 5' ends - are added to each
fragment and used for several reactions to make the fragments finally to single strands.
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Hundreds of thousands of single stranded DNA fragments will be bound to beads via adapters. These will form micro reactors which enclose one bead and one DNA fragment with oil-water emulsion.
These will be amplified to several millions of copies per bead by emPCR.
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After the amplification reaction, oil-water emulsion will be destroyed in order to obtain the beads. These beads are collected and enriched afterwards,
and loaded onto a plate with gaskets for sequencing.
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Sequencing Analysis
Using extension reaction by DNA polymerase just same as normal PCR, bases will be loaded in a fixed order of T -> A -> G -> C.
Sulfurylase generates ATP using pyrophoric acid, which is made in polymerase extension reaction, as a substrate. Luciferase reacts to generate a light
signal using this ATP and Luciferin as substrates. This chemiluminescent signal is recorded by the CCD camera.
ATP used for luminous reaction is degraded to dNMP by Apyrase and ceases to generate a light signal.
dNTPs not used for polymerization is degraded to dNMP by Apyrase and are not used for the next extension reaction.
1. Capture of raw data photo
2. Extraction of each well's data
3. Conversion to bar graph
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Sequencing analysis principle is same as single read analysis.