Bioinformatics Lab lecture 2022

1. Sequencing
MICROBIO 590B Bioinformatics Lab: Bacterial Genomics
Professor Kristen DeAngelis
UMass Amherst
Fall 2022
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2. Lecture Learning Goals
• Define first, next (aka second) and third genera5on sequencing.
• Explain the key innova5on in development of each type of
sequencing, and contrast how sequencing works for each.
• Explain how Sanger sequencing works.
• Explain how sequencing by synthesis (Illumina technology) works.
• Explain how nanopore (long read) sequencing works.
• Describe the benefits and tradeoffs of sequence length, output, and
fidelity for each kind of sequencing technology.
• List some advantages for portable sequencing technology like
MinION.
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3. DNA structure
• The structure of DNA was solved in 1953.
• Watson and Crick identified the role of DNA
in heredity, but Linus Pauling, Watson and
Crick, had an erroneous triple helix model,
and then, a model with the chains inside
and the bases pointing outwards.
• Franklin’s identification of the space group
for DNA crystals revealed to Crick that the
two DNA strands were antiparallel, and
proved that the phosphates were on the
outside of the helix.
• Photo 51 shows the X-ray diffraction pattern
of DNA taken by Raymond Gosling, a
graduate student in Rosalind Franklin’s lab.
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Pencil sketch of the DNA double
helix by Francis Crick in 1953
Photo 51, Gosling &
Franklin, 1952

4. A Brief History of Sequencing Technology
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5. Evolution of Sequencing
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Arvind Varsani, adapted from Stéphane Le Crom

6. Milestones in genome assembly
6
Giani et al., Computational and Structural Biotechnology Journal 2020

7. Outline
• Sanger Sequencing
• Next Genera5on Sequencing
• Illumina
• Third Genera5on Sequencing
• PacBio
• Oxford Nanopore
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8. Sanger Sequencing
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PCR sequence
amplification
Reaction with terminator
nucleotides
Separate variable sized
oligonucleotides using
capillary electrophoresis
Use the chromatograph
for base calling
Trim and QC sequences
A row of DNA sequencing machines; from Flickr

9. Sanger
Sequencing
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10. • Chain-termination di-deoxy
sequencing
• These ddNTPs lack a 3’-OH
group that is required for chain
elongation
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Sanger Sequencing

11. 11
Sanger Sequencing

12. Sanger Sequencing
• For each DNA sequence, four
separate reactions are run that
include a mix of dNTPs and some
ddATP, ddTTP, ddCTP or ddGTP
• The mix must have only ~1% ddNTP to
allow for enough fragments to be
produced while still transcribing the
complete sequence
• This is called sequencing by synthesis:
the DNA polymerase makes a copy of
the template, and when it encounters
the ddNTP, the fragment synthesis is
terminated.
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Sanger et al., PNAS 1977

13. Sanger Sequencing
• Fluorescent terminators allow for
automation
• Capillary electrophoresis is a method
of electrokinetic separation, just like
gel electrophoresis
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14. Sanger Sequencing
• Many free programs are available to analyze these chromatographs
• 4Peaks (Mac), Sequencher (Mac, PC), Geneious (Mac, PC, Linux)
• Use these programs to:
• Remove the primer sequences
• Remove the poor sequence at the beginning and end of the reads
• Make or correct base calls manually & check for multiple templates
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15. Outline
• Sanger Sequencing
• Next Generation Sequencing
• Illumina
• Third Generation Sequencing
• PacBio
• Oxford Nanopore
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16. 16
Next Generation Sequencing
hGps://www.biochain.com/general/from-sanger-to-next-generaNon-sequencing/

17. 17
Next Generation Sequencing
genome.gov/sequencingcosts

18. Next Generation Sequencing
• Three new technologies helped to usher in a new generation of
sequencing : (1) emulsion PCR
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(a) DNA molecules being clonally amplified in an emulsion PCR (emPCR). Adapter ligation and PCR produces DNA libraries with appropriate 5ʹ and 3ʹ ends, which
can then be made single stranded and immobilized onto individual suitably oligonucleotide-tagged microbeads. Bead-DNA conjugates can then be emulsified
using aqueous amplification reagents in oil, ideally producing emulsion droplets containing only one bead (illustrated in the two leftmost droplets, with different
molecules indicated in different colours). Clonal amplification then occurs during the emPCR as each template DNA is physically separate from all others, with
daughter molecules remaining bound to the microbeads. This is the conceptual basis underlying sequencing in 454, Ion Torrent and polony sequencing protocols.
Heather & Chain, Genomics 2016

19. Next Generation Sequencing
• Three new technologies helped to usher in a new generation of
sequencing : (2) bridge amplification
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Heather & Chain, Genomics 2016
(b) Bridge amplification to produce clusters of clonal DNA populations in a planar solid-phase PCR reaction, as occurs in Solexa/Illumina sequencing. Single-
stranded DNA with terminating sequences complementary to the two lawn-oligos will anneal when washed over the flow-cell, and during isothermal PCR will
replicate in a confined area, bending over to prime at neighbouring sites, producing a local cluster of identical molecules.

20. Next Generation Sequencing
• Three new technologies helped to usher in a new generation of
sequencing : (3) picotiter plates and flow-cells
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Heather & Chain, Genomics 2016
(c) and (d) demonstrate how these two different forms of clonally-amplified sequences can then be read in a highly parallelized manner: emPCR-produced
microbeads can be washed over a picotiter plate, containing wells large enough to fit only one bead (c). DNA polymerase can then be added to the wells, and
each nucleotide can be washed over in turn, and dNTP incorporation monitored (e.g. via pyrophosphate or hydrogen ion release). Flow-cell bound clusters
produced via bridge amplification (d) can be visualized by detecting fluorescent reversible-terminator nucleotides at the ends of a proceeding extension reaction,
requiring cycle-by-cycle measurements and removal of terminators.

21. Next Generation Sequencing
= Massively Parallel Sequencing
21
Metker (2010) Nat Rev Genetics

22. Illumina Sequencing
• Sequencing by synthesis
• also how Sanger works
• Steps
1. Library prep
2. Sequencing
3. Data analysis
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23. Illumina Sequencing
1. Library prep
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24. Illumina Sequencing
1. Library prep
2. Sequencing
3. Data analysis
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https://www.neb.com/applications/ngs-sample-prep-and-target-enrichment/illumina-library-preparation

25. Illumina Sequencing
1. Library prep
2. Sequencing
3. Data analysis
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26. Illumina Sequencing
1. Library prep
2. Sequencing
3. Data analysis
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https://www.youtube.com/watch?v=womKfikWlxM

27. Illumina Sequencing
1. Library prep
2. Sequencing
3. Data analysis
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28. Illumina Sequencing
1. Library prep
2. Sequencing
3. Data analysis
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• DNA-seq Genomic DNA
• RNA-seq RNA
• Ribo-seq Ribosomal Profiling
• miRNA-seq Micro RNA profiling
• ChIP-Seq DNA-protein interactions
• BS-seq Methylation via Bisulfite
• RAD-seq Restriction Site Associated DNA Markers
• Hi-C-seq Chromatin Interaction Profiling

29. Outline
• Sanger Sequencing
• Next Generation Sequencing
• Illumina
• Third Generation Sequencing
• PacBio
• Oxford Nanopore
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30. Sanger, Next Gen, and Third Gen Sequencers
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https://flxlexblog.wordpress.com/2016/07/08/developments-in-high-throughput-sequencing-july-2016-edition/

31. PacBio long-read sequencing
• PacBio developed the platform of single molecule real time (SMRT) sequencing
• Signals are fluorescent light emitted from each nucleotide incorporated by a DNA
polymerase bound to the bottom of each microtiter well.
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32. PacBio long-read sequencing
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Eid et al., Science 2009
• Single-molecule real-time sequencing utilizes a zero-mode waveguide (ZMW).
• A single DNA polymerase enzyme is affixed at the bottom of each ZMW with a
single molecule of DNA as a template.

33. DNA base modifications
• Both PacBio and Oxford Nanopore sequencing detect modified bases
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34. Oxford Nanopore Sequencing
• Animation showing a DNA strand being fed through a nanopore
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35. Oxford Nanopore Sequencing
• Animation showing signal being read from DNA strand as it passes
through a nanopore
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36. Oxford Nanopore Sequencing
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Zhang et al., 2019 https://doi.org/10.1101/747113
• Oxford Nanopore sequencing is sensitive to detecting modified bases

37. Oxford Nanopore Sequencing is portable!
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American astronaut Kate
Rubins with a MinION
sequencer on the
International Space
Station in August 2016
Castro-Wallace et al., 2017

38. Oxford Nanopore Sequencing is portable!
• …
38
https://nanoporetech.com/about-us/news/field-kit-launched-oxford-nanopore-enabling-shipping-and-storage-sequencing

39. Oxford Nanopore Sequencing is portable!
• allow farmers to identify fungicide resistance
genotypes in pathogen populations & make
decisions about fungicide spraying
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#sequencing4farmers, twiiter Dr. Laura Boykin-Okalebo @laura_boykin, @maclaranlee

40. Third generation sequencing
= long read sequencing
• Uses native DNA
without fragmentation,
so DNA quality is
paramount
• Capable of producing
very long reads
• Throughput is much
lower than 2nd (next)
gen sequencing
40
Patterson, Wongsurawat, Rodriguez.
Medical research archives 8, no. 2 (2020).

41. Lecture Learning Goals
• Define first, next (aka second) and third generation sequencing.
• Explain the key innovation in development of each type of
sequencing, and contrast how sequencing works for each.
• Explain how Sanger sequencing works.
• Explain how sequencing by synthesis (Illumina technology) works.
• Explain how nanopore (long read) sequencing works.
• Describe the benefits and tradeoffs of sequence length, output, and
fidelity for each kind of sequencing technology.
• List some advantages for portable sequencing technology like
MinION.
41

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