sequencing technologies - andrew-michaelson.com · objectives •understand sanger sequencing...

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.

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

http://classroom.sdmesa.edu/eschmid/Lab12 1.jpg

https://www.premedhq.com/gel-electrophoresis-and-southern-blotting

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


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


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


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


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.


Next-Gen Sequencing

Results

4-mer

3-mer

2-mer

1-mer

A

T

G

C


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


http://masscience.com/wp-content/uploads/2015/06/204g_expression-confirmation_blast-EST.jpg


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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