Embed Size (px)
Citation preview
Next Generation Sequencing
454-Pyrosequencing
Sample Input &
Fragmentation
• First the Sample of interest is
obtained
• Library is fragmented
• Sonication
• Nebulization
Image from http://454.com/products/technology.asp
Library Preparation
• Adapter ligated
• Then denatured
• 95 degrees Celsius
Image from http://454.com/products/technology.asp
One Fragment = One
Bead
• Anneal ssDNA to an excess of
DNA capture beads
• fragment:bead complexes mixed with
emulsion oil
• emulsion formation
Image from http://454.com/products/technology.asp
ePCR: Emulsion PCR
Amplification
• Microreactors – fragment:bead
complex and PCR reagents
• DNA polymerase
• Primers
• Buffers
• dNTPs
• Clonal amplification occurs inside
• microreactors
Image from http://454.com/products/technology.asp
Sequencing: One
Bead = One Read
• micelles broken and enriched for DNA-
positive beads.
• Loaded into a picotiter plate
• Enzyme beads loaded
Pyrosequencing
Chemistry
• Cycles the four bases (ATGC) are
sequentially washed over the PicoTiterPlate.
• inorganic pyrophosphate starts a
chemical cascade.
• CCD camera captures the light signal
given off
Image from http://454.com/products/technology.asp
Data Processing &
Analysis
• Run Time Analysis
• Image acquisition
• Image processing
• Signal processing
• Post-run Processing
• Assembly
• Mapping
• Amplicon Variant Analysis
Image from http://454.com/products/technology.asp
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.
• 454 Sequencing. The Technology: System Workflow.
http://454.com/products/technology.asp. Access verified July 30, 2014.
• PYROSEQUENCING: Genome Sequencing Utilizing Light-Emitting Luciferase
and PCR-Reaction-Mixture-in-Oil Emulsion. Mr. Meir Shachar and Dr. Edwin
Ginés-Candelaria. Available at
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&ua
ct=8&ved=0CEEQFjAB&url=http%3A%2F%2Fwcis.mdc.edu%2Fegines%2FBSC294
3L_THE%2520BIOSCIENCE%2520INTERNSHIP%2FNEXT_GENERATION%2520
SEQUENCING%2FPUBLICATIONS%2F454%2FPOWER%2520POINT%2FPYROS
EQUENCING.ppt&ei=mO3gU7e3MY-
3yATKvILoAw&usg=AFQjCNFUTJOwRiQVfr4QOpRwtEoW7E4Gow&sig2=5E5kc
HvdziwacvOOtvRxow. Access verified Aug 5, 2014.
Next Generation Sequencing
Illumina Sequencing
