McGill University and Genome Quebec Innovation Centre
at Génome Québec
Montreal, Quebec, Canada
2 Total Orders0 Endorsements
CEN4GEN Institute for Genomics and Molecular Diagnostics
Edmonton, Alberta, Canada
0 Total Orders0 Endorsements
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PacBio SMRT sequencing uses sequence by synthesis (SBS) methodology, but is able to visualize the incorporation of nucleotides by a polymerase in real time. The reaction takes place in a microscopic glass-bottomed cylindrical chamber termed a zero-mode waveguide (ZMW); 150,000 ZMWs are patterned on each SMRT cell. The polymerase is tethered to the glass bottom and the well is flooded with nucleotides tagged on the 3â€™ phosphate group with an identifying fluorophore. When a nucleotide is incorporated by the polymerase, its signal is detected through the glass bottom before it is cleaved off. Thus, nucleotide identity can be read as the polymerase processes at its natural speed. SMRT sequencing generates extremely long reads averaging 3,000 to 5,000 base pairs, which is extremely useful for de novo genome assembly.
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