Editing BioMicroCenter:PacBio (section)

== [http://www.pacb.com/products/smrt-technology/ HOW SINGLE MOLECULE SEQUENCING WORKS ]==
The Revio works by detecting DNA replication in real time. Fluors are attached to the gamma-phosphate of the nucleotides. Ideally, a PacBio library with a single polymerase is loaded into each well. When a fluorescent nucleotide enters the active site, it can be detected by the sequencer. Basepaired nucleotides have a longer dwell time allowing them to be detected. <BR><BR>
The Revio is able to monitor ~10 M incorporations occurring simultaneously on a SMRTcell. Each run creates a “movie” of the incorporation of bases, collecting data on the intensity of each fluorophore in each well over time. On board computation converts this movie into basecall files. <BR><br>

[[IMAGE:CCS-demo.jpg|thumb|left|400px|Diagram of CCS analysis]]

Using an analysis mode called Circular Consensus Sequencing, high fidelity (HiFi) subreads can be assembled when libraries are sequenced end to end in rolling fashion multiple times.  In CCS, a 2 kbp amplicon can easily be sequenced over 10 times end to end over the course of a 24 hr movie. Rare lengths of 175 kbp have been seen, but it is more typical to see read lengths 50-70 kbp:  the longest reads are typically outliers since the average subread length is typically <50 kbp.

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== EXPECTED RESULTS ==
TYPICAL RESULTS FROM SMRTCELLS:
* 20kb fragments – 50,000 active sites – 400Mbp sequence
* 2kb fragments – 80,000 active sites – 500Mbp sequence

Typical Read Lengths
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!Genomic!!Amplicon (<2 kb)
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|[[IMAGE:PacBioSequelTypicalv2.jpg|thumb|left|400px|Genomic DNA prepared with Template Kit 1.0 and BluePippin on Sequel 2.1 Chemistry]]||[[IMAGE:PacBioSequelTypicalv2-amp.jpg|thumb|left|400px|PCR Amplicon prepared with Express Template Kit 2.0 on Sequel 3.0 Chemistry]]
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|Longest Inserts of around 60 kbp in CLR||Shorter Inserts mean higher Quality Scores in CCS
|}
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