Next Generation Sequencing

Single Molecule Real Time Method

Overview

• Uses actual DNA polymerase to sequence

• Enzymatic speed

• fluorescent probe not to the nucleotide

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

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/

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

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

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

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

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.

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/

Next Generation Sequencing

Ion Torrent Method