presentacao de next generation sequencing

1. Next Generation
Sequencing
by Dr. Kamila A Meissner

2. 1st Generation Sequencing
• Sanger Chain Termination
• 1977
• DNA-Polymerase
• Di-deoxynucleotides with
fluorescence dye
• Primer depended
• Capillary based automated
electrophoreses
• Routine sequencing
• Maxam-Gilbert Sequencing
• Almost not used today
https://www.onlinebiologynotes.com/sangers-method-gene-sequencing/

3. 1st Generation Sequencing
• Sanger Chain Termination
• 1977
• DNA-Polymerase
• Di-deoxynucleotides with
fluorescence dye
• Primer depended
• Capillary based automated
electrophoreses
• Routine sequencing
• Maxam-Gilbert Sequencing
• Usage of radioactive 32P
• Chemical termination
• Almost not used today
https://www.onlinebiologynotes.com/sangers-method-gene-sequencing/

4. NewGeneration Sequencing NGS
• High throughput sequencing
• Reduced sample size
• >1000 sequences simultaneously
• Lower costs
• Less time
• No cloning
• Revolutionizing personalized medicine, genetic diseases and diagnostic
Illumina MiSeq Illumina HiSeq Roche 454 Life Technologies SOLiD5500 QIAGEN GeneReader

5. NewGeneration Sequencing NGS
Illumina MiSeq Illumina HiSeq Roche 454 Life Technologies SOLiD5500 QIAGEN GeneReader
Cojocneanu et al. 2015

6. Sample Preparation
• Extraction, fragmentation and adaptor ligation
• Emulsion PCR
• DNA fragments flanked with adaptors
• Primer linked on bead surface
• PCR in water drop in oil
• Bridge PCR
• DNA fragments flanked with adaptors
• Primers complementary to adaptors
• Isothermal amplification
Leong et al. 2014

7. Pyrosequencing
• Bioluminescence
method
• Adding one dNTP at a
time
• Measures release of
inorganic pyrophosphate
• Enzymatic reactions
emitting light
Whiteford 2007

8. Sequencing by Synthesis
• Cyclic Reversible termination
• Nucleotides containing
terminator, fluorescence dye
and cleavage site
• All dNTP used at the same time
• Washing and cleaving steps
Guo et al. 2009

9. Sequencing by Ligation
• No DNA-Polymerase
• 16 octamer oligo probes with
fluorescent dye
• DNA-Ligase reaction
• Cleavage of last 3 dNTPs and dye
• Denaturation and repeating the ligation
process shifted by one position
Voelkerding et al. 2009

10. Ion Semiconductor Sequencing
• Similar to Pyrosequencing
• Measurement of released hydrogen
Robinson 2011

11. NGS Applications
NGS
ChIPseq
Whole
genome seq
Clinical
detection
Metagenomics
And much
much more....
Molecular
diagnostics
Mutation
discovery
SingleCell seq
RNAseq

12. Comparison of NGS
Method Pyrosequencing (454)
Sequencing by
Synthesis (Illumina)
Sequencing by
Ligation (SOLiD)
Ion
Semiconductor
Chain termination
(Sanger)
Read length 700 bp 50-250 bp rd. 100 bp 200bp 400-900 bp
Accuracy 99.9% 98% 99.9% 98% 99.9%
Reads per run 1 million Up to 3 billion Up to 1.5 billion
Up to 5 million
Time per run 24 h 1 to 10 days 1 to 2 weeks 2 h 20 min to 3 h
Cost per 1 mil
bases
$10 $0.05 to $0.15 $0.13 $1 $2400
Advantagens Long read size and fast High sequence yield Low cost
Long reads
Different applications
Disvantagens
Expensive and
homopolymere errors
Can get very
expensive
Very slow
Homopolymere
errors
Expensive and difficult
for large sequencing
projects

13. Thank you
Questions.....

14. References
• Leong, Ivone & Skinner, Jonathan & Love, Donald. (2014). Application of Massively Parallel Sequencing in the
Clinical Diagnostic Testing of Inherited Cardiac Conditions. Medical Sciences. 2. 98-126.
10.3390/medsci2020098.
• https://www.onlinebiologynotes.com/sangers-method-gene-sequencing/
• Cojocneanu, Roxana & Pop, Laura & Jurj, Ancuta & Raduly, Lajos & Dumitrascu, Dan & Dragos, Nicolae &
Berindan - Neagoe, Ioana. (2015). Next generation sequencing applications for breast cancer research. Clujul
Medical. 88. 278. 10.15386/cjmed-486.
• Whiteford, Nava. (2007). UNIVERSITY OF SOUTHAMPTON String Matching in DNA Sequences: Implications
for Short Read Sequencing and Repeat Visualisation.
• JIA GUO LIN YU, NICHOLAS J. TURRO, AND JINGYUE JU. An Integrated System for DNA Sequencing by
Synthesis Using Novel Nucleotide Analogues (2009)
• V Voelkerding, Karl & Dames, Shale & D Durtschi, Jacob. (2009). Next-Generation Sequencing: From Basic
Research to Diagnostics. Clinical chemistry. 55. 641-58. 10.1373/clinchem.2008.112789.
• Keith Robison Semiconductors charge into sequencing Nature Biotechnology volume 29, pages 805–807
(2011)
• Escalante, Ana & Jardón-Barbolla, Lev & Ramírez-Barahona, Santiago & Eguiarte, Luís. (2014). The study of
biodiversity in the era of massive sequencing El estudio de la biodiversidad en la era de la secuenciación
masiva. Revista Mexicana de Biodiversidad. 85. 1249-1264. 10.7550/rmb.43498.

15. Single Molecule Real Time Sequencing (SMRT)
• zero mode waveguide (ZMW)
Escalante et al. 2014