1. Sequencing Technologies

2. Objectives Understand Sanger sequencing (first generation)
Understand Next Gen Sequencing
Understand 3rd Generation Nanopore sequencing
Review paper

3. Sequencing Timeline

4. Sequencing Timeline

5. Cost of sequencing the human genome

6. Nucleic acid hybridization is the basis of sequencing technologies
Complementary Base Pairing
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

7. Modern Sequencing is Automated
(a) Standard sequencing machine
Figure 20.2 DNA sequencing machines
(b) Next-generation sequencing machines
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

8. Mardis, Elaine R. "DNA sequencing technologies: 2006-2016
Mardis, Elaine R. "DNA sequencing technologies: " Nature Protocols 12.2 (2017):

9. Mardis, Elaine R. "DNA sequencing technologies: 2006-2016
Mardis, Elaine R. "DNA sequencing technologies: " Nature Protocols 12.2 (2017):

10. Overview of Sanger Seqeuncing Steps
DNA is denatured
Primer is annealed to DNA
DNA Polymerase uses a radiolabeled deoxynucleotide triphosphates (normal dNTPs) and a few chain-terminating dideoxynucleotide triphosphates (ddNTPs) to build strand.
DNA run on Polyacrylamide gels
DNA read via x-ray

11. DNA Sequencing
Polyacrylamide gel electrophoresis separates ssDNA molecules that differ in length by just one nucleotide
Molecules are labelled with a radioactive protein or radioactive isotope, visualized by autoradiography producing a banding pattern

12. Sanger Sequencing Technique DNA (template strand) Primer DNA3′ T G 5′ C T G
A C A 5′
DNA
polymerase
Figure 20.3a Research method: dideoxy chain termination method for sequencing DNA (part 1) 3′
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

13. Deoxyribo- nucleotides
Sanger Sequencing
Technique
DNA
(template strand)
Primer
Deoxyribo- nucleotides
Dideoxyribonucleotides (fluorescently tagged) 3′ T G 5′ C T G
A C A
dATP
ddATP 5′
dCTP
ddCTP
DNA
polymerase
dTTP
ddTTP
dGTP
ddGTP
Figure 20.3a Research method: dideoxy chain termination method for sequencing DNA (part 1) P P P P P P G G 3′
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

14. Deoxyribo- nucleotides
Sanger Sequencing
Technique
DNA
(template strand)
Primer
Deoxyribo- nucleotides
Dideoxyribonucleotides (fluorescently tagged) 3′ T G 5′ C T G
A C A
dATP
ddATP 5′
dCTP
ddCTP
DNA
polymerase
dTTP
ddTTP
dGTP
ddGTP
Figure 20.3a Research method: dideoxy chain termination method for sequencing DNA (part 1) P P P P P P G G 3′
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

15. Sanger Sequencing Technique DNA (template strand) Labeled strands 5′3′ C T G
A C A dd G A C T dd A C T G dd C T G A dd T G A dd G A dd A G dd A G A G C T dd 3′ G C T dd C T G C T G C T G C T G C T G C T G C T G
Figure 20.3b Research method: dideoxy chain termination method for sequencing DNA (part 2) 5′ 3′ 5′
Shortest
Longest
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

16. Sanger Sequencing Technique Direction of movement of strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
Results
Last nucleotide of longest labeled strand
Figure 20.3c Research method: dideoxy chain termination method for sequencing DNA (part 3) G A C T G A C
Last nucleotide of shortest labeled strand
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

17. Human Genome Shotgun Sequencing
Bacterial Artificial Chromosome (BAC)
Bacterial Artificial Chromosome (BAC)

18. Next-Gen Sequencing Second Generation Sequencing Technique1
Genomic DNA is fragmented. 2
Each fragment is isolated with a bead. 3
Using PCR, 106 copies of each fragment are made, each attached to the bead by 5′ end. 4
The bead is placed into a well with DNA polymerases and primers.
Figure 20.4a Research method: next-generation sequencing (part 1)
Template strand of DNA 3′ 5′ 3′ 5′
Primer A T G C 5
A solution of each of the four nucleotides is added to all wells and then washed off. The entire process is then repeated.
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

19. Next-Gen Sequencing TechniqueA T G C A T G C
Template strand of DNA C C C C A A
dTTP A
dATP A T T G G T A
PPi T A
DNA polymerase G C G C G C G C A G
Figure 20.4b Research method: next-generation sequencing (part 2)
Primer A G T A T A 6 7
If a nucleotide is joined to a growing strand, PPi is released, causing a flash
of light that is recorded.
If a nucleotide is not complementary to the next template base, no PPi is released, and no flash of light is recorded.
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

20. Next-Gen Sequencing Technique 8A T G C A T G C C A T G C A T G
dCTP
dGTP C
PPi A
Figure 20.4c Research method: next-generation sequencing (part 3) 8
The process is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence.
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

21. Next-Gen Sequencing Results A 4-mer T G 3-mer C 2-mer 1-mer
Figure 20.4d Research method: next-generation sequencing (part 4)
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)

22. Figure 1
454 Workflow: library construction ligates 454-specific adapters to DNA fragments and couples amplification beads with DNA in an emulsion PCR to amplify fragments before sequencing. The beads are loaded into the picotiter plate (PTP). The bottom panel illustrates the pyrosequencing reaction that occurs on nucleotide incorporation to report sequencing by synthesis.
Trends in Genetics  , DOI: ( /j.tig )

23. Nanopore Sequencing

24. https://www. slideshare

25. Multiple sequence alignment
Novel BHD missense mutation (K508R). (A) Genomic sequence of control. (B) Genomic sequence of BHDS patient with the c.1978A>G (K508R) mutation. (C) Multiple sequence alignment. The amino acid residues are coloured according to their chemical properties. (D) Degree of conservation. The numerical index reflects the degree of conservation of the physical–chemical properties in the alignment. Star (*) indicates amino acids 100% identical in the alignment (highest score). The next most conserved group is composed of substitutions in amino acids with the same physical chemical class.

26. Multiple sequence alignment
Interactive Structure based Sequences Alignment Program Strap
Interactive Structure based  Sequences Alignment Program Strap

27. Multiple sequence alignment
BLAST: Basic Local Alignment Search Tool

28. High throughput Technology Qiagen Gene Reader, sequencing by sequence (SBS)
Automation
Bioinformatics pipeline
Identification of mutations and best therapy
This is a complete solution

29. Challenges of NGS

30. https://www. slideshare

31. Summary: High throughput Technology

32. Worksheet 2!