1.
Next Generation Sequencing
Single Molecule Real Time Method

2.
Overview
• Uses actual DNA polymerase to sequence
• Enzymatic speed
• fluorescent probe not to the nucleotide

3.
Step 1
• A complex of a DNA template and active
polymerase is immobilized at the bottom of
the ZMW
Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php

4.
Step 2
• Phospho-linked nucleotides are introduced
into the chamber of the ZMW.
Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php
Image found at http://www.pacificbiosciences.com/products/smrt-technology/

5.
Step 3
• Each of the four nucleotides is labeled with a
different colored fluorophore and loaded.
Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php
Image found at http://www.umich.edu/~caparray/products/ngs/pacbio

6.
Step 4
• As a base is held in the detection volume, a
light pulse is produced.
Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php

7.
Step 5
• Nucleotides held by the polymerase prior to
incorporation emit an extended signal that
identifies the base being incorporated.
Image found at http://www.omixon.com/the-basics-of-next-generation-sequencing/
Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php

8.
Step 6
• This process occurs in parallel in up to
thousands of ZMWs that make up the SMRT
cell.
Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php

9.
Work Cited
• Ambry Genetics. Making Sense of NextGent Sequencing. Kelly Gonzalez, MS, CGC,
and Senior Manager of Clinical Genomics. Available at
http://www.ambrygen.com/sites/default/files/pdfs/NERRG_4-10-
12_Making_Sense_of_NetGen_Sequencing_KG(3).pdf. Access verified May 21,
2014.
• Omixon. Allen Van Deynze, 2010; Solanaceae Coordinated Agricultural Project. Next
Generation Sequencing. Available at http://www.omixon.com/the-basics-of-next-
generation-sequencing/. Access verified May 21, 2014.
• DNA Sequencing, Sanger and Next-Generation Sequencing. Omics eBooks Group.
(2014). Yao Fei, Genome Institute of Singapore. Available at
http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-
sequencing-sanger-and-nextgeneration-sequencing.php Access verified Aug 5,
2014.
• SMRT TECHNOLOGY. Pacific Biosciences. (2014). Available at
http://www.pacificbiosciences.com/products/smrt-technology/ Access verified Aug 5,
2014.
• PacBio RS II Sequencing. Microarray Core Facility. (2012). Available at
http://www.umich.edu/~caparray/products/ngs/pacbio/ Access verified Aug 5, 2014.
• DNA SEQUENCING/GENOMICS: Toward personalized medicine: 3G DNA
sequencing. (March 2010). Stephen Turner. BioOptics. Available at
http://www.bioopticsworld.com/articles/print/volume-3/issue-3/features/dna-
sequencing-genomics.html Access verified Aug 5, 2014.

10.
Recommended Videos
SMRT TECHNOLOGY
http://www.pacificbiosciences.com/produc
ts/smrt-technology/
Introduction to SMRT Sequencing
http://article.wn.com/view/2014/05/14/Sin
gle_Molecule_RealTime_SMRT_DNA_S
equencing_Technology_From_g/

11.
Next Generation Sequencing
Ion Torrent Method