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Sequencing Technologies
Objectives
• Understand Sanger sequencing (first generation)
• Understand Next Gen Sequencing
• Understand 3rd Generation Nanopore sequencing
• Review paper
Sequencing Timeline
http://www.yourgenome.org/facts/timeline-organisms-that-have-had-their-genomes-sequenced
http://genomicsforeveryone.org/week-1-genetics-and-public-health-genomics/
Sequencing Timeline
Cost of sequencing the human
genome
https://www.genome.gov/sequencingcostsdata/
Nucleic acid hybridization is the
basis of sequencing technologies
Complementary Base PairingReece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)
(a) Standard sequencing machine
(b) Next-generation sequencing machines
Modern Sequencing is Automated
Mardis, Elaine R. "DNA sequencing technologies: 2006-2016." Nature Protocols 12.2 (2017): 213-218.
Mardis, Elaine R. "DNA sequencing technologies: 2006-2016." Nature Protocols 12.2 (2017): 213-218.
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 is denatured
• DNA run on Polyacrylamide gels
• DNA read via x-ray
http://www.csus.edu/indiv/r/rogersa/bio181/seqsanger.pdf
DNA Sequencing
http://classroom.sdmesa.edu/eschmid/Lab12%201.jpghttps://www.premedhq.com/gel-electrophoresis-and-southern-blotting
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
DNA
(template strand)
Primer
Technique
DNA
polymerase
5′
5′
3′
3′
CTGACTTCGACAA
TGTT
Sanger Sequencing
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014) http://www.csus.edu/indiv/r/rogersa/bio181/seqsanger.pdf
DNA
(template strand)
Primer Deoxyribo-
nucleotidesDideoxyribonucleotides
(fluorescently tagged)
Technique
DNA
polymerase
5′
5′
3′
3′
CTGACTTCGACAA
TGTT
dATP ddATP
dCTP ddCTP
dTTP ddTTP
dGTP ddGTP
P P P P P PG G
Sanger Sequencing
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)
DNA
(template strand)
Primer Deoxyribo-
nucleotidesDideoxyribonucleotides
(fluorescently tagged)
Technique
DNA
polymerase
5′
5′
3′
3′
CTGACTTCGACAA
TGTT
dATP ddATP
dCTP ddCTP
dTTP ddTTP
dGTP ddGTP
P P P P P PG G
Sanger Sequencing
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)
DNA (template
strand)
Labeled strands
Shortest Longest
5′
5′ 5′3′
3′
3′CTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
Sanger Sequencing
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)
Direction
of movement
of strands
Longest labeled strand
Detector
LaserShortest labeled strand
Results
Last nucleotide
of longest
labeled strand
Last nucleotide
of shortest
labeled strand
GACTGAAGC
Technique
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)
Sanger Sequencing
Human Genome Shotgun Sequencing
http://www.genome.ou.edu/3653/3653-101705.htmlhttps://www.ndsu.edu/pubweb/~mcclean/plsc411/Genome%20Sequencing%20Complete.pdf
Bacterial Artificial Chromosome (BAC)
Technique
Genomic DNA is fragmented.
Each fragment is isolated with
a bead.
Using PCR, 106 copies of each
fragment are made, each attached
to the bead by 5′ end.
The bead is placed into a well with
DNA polymerases and primers.
4
Template strand
of DNA
Primer3′
A
3′
5′ 5′T G C
3
2
1
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)
Next-Gen
Sequencing
Second Generation Sequencing
6 7If a nucleotide is joined to a growing strand, PPi is released, causing a flashof light that is recorded.
If a nucleotide is notcomplementary to thenext template base,no PPi is released, andno flash of light is recorded.
Template
strand
of DNA
Primer
DNA
polymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
Technique
A T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
G
A
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)
Next-Gen Sequencing
8
Technique
A T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
G
A AC
dGTP dCTP
PPi
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)
Next-Gen Sequencing
Results
4-mer
3-mer
2-mer
1-mer
A
T
G
C
Reece, J. B. et al. Campbell Biology (New York; Pearson Education Inc., 2014)
Next-Gen Sequencing
Figure 1
Trends in Genetics 2008 24, 133-141DOI: (10.1016/j.tig.2007.12.007)
http://www.cell.com/trends/genetics/fulltext/S0168-9525(08)00023-1
http://www2.technologyreview.com/news/427677/nanopore-sequencing/
Nanopore Sequencinghttps://www.nextgenerationsequencing.info/ngs-products/ngs-technologies/oxford-nanopore-technologies
https://www.slideshare.net/AnneliesHaegeman/annelies-haegeman-ngs-basic-principles-and-sequencing-platforms
http://jmg.bmj.com/content/45/6/3210
Multiple sequence alignment
https://www.mathworks.com/help/bioinfo/ug/viewing-and-aligning-multiple-sequences.html?requestedDomain=www.mathworks.com
Interactive Structure based Sequences Alignment Program Strap
http://www.bioinformatics.org/strap/index2.html
Multiple sequence alignment
http://masscience.com/wp-content/uploads/2015/06/204g_expression-confirmation_blast-EST.jpg
Multiple sequence alignment
High throughput TechnologyQiagen Gene Reader, sequencing by
sequence (SBS)• Automation
• Bioinformatics pipeline
• Identification of mutations and best therapy
https://www.youtube.com/watch?v=HQhw5Ihp8IAhttps://www.qiagen.com/us/products/ngs/mdx-ngs-genereader/
https://www.slideshare.net/ueb52/introduction-to-next-generation-sequencing-v2?next_slideshow=1
Challenges of NGS
https://www.slideshare.net/ueb52/introduction-to-next-generation-sequencing-v2?next_slideshow=1
Summary: High throughput Technology
https://www.qiagen.com/us/products/ngs/mdx-ngs-genereader/
Worksheet 2!
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