It deals with principles, procedure and application of Ion torrent and SOLiD Sequencing methods.

1. Ion Torrent (Proton/PGM) Sequencing
and
SOLiD Sequencing
Dr. Fikre Zeru (DVM, MSc,
Assistant Professor at Mekelle university
PhD fellow at AAU, IOB, SIDA project, Uppsala University)
Addis Ababa,
March, 2019

2. Introduction to Sequencing
• Genome sequencing is figuring out the order of DNA
nucleotides
– to read the genetic information found in the DNA
• In 1977, Frederick Sanger developed DNA sequencing
technology known as Sanger sequencing
– It was laborious and radioactive materials were required.
• The automatic sequencing instruments (namely AB370)
– which made the sequencing faster and more accurate.
– became the main tools for the completion of human genome
project in 2001.
• The human genome project greatly stimulated the
development of powerful novel sequencing instrument to
increase speed and accuracy, while simultaneously reducing
cost and manpower……………….NGS

4. NGS
• NGS, also known as high-throughput sequencing, including:
– The first NGS technology released in 2005 was the pyrosequencing method by
454 Life Sciences (now Roche).
– One year later, the Solexa/Illumina sequencing platform was commercialized
(Illumina acquired Solexa) .
– The third technology to be released was Sequencing by Oligo Ligation
Detection (SOLiD) by Applied Biosystems (now Life Technologies) in 2007.
– In 2010, Ion Torrent (now Life Technologies) released the Personal Genome
Machine (PGM).
• The NGS technologies are different from the Sanger method in
aspects of massively parallel analysis, high throughput, and reduced
cost.

5. CONT’D
• NGS has significantly decreased the time and cost
required for producing large quantities of sequence
data.
• These technologies have increased the feasibility of
– analyzing genomes and transcriptomes,
– identifying new genes and genetic markers, and
– investigating genome homology,
– differential gene expression, and epistasis.

6. Ion torrent: Proton / PGM sequencing
• Ion Semiconductor Sequencing is a method of NGS based
on the detection of H+ ions that are released during the
polymerization of DNA.
– It is sequencing by synthesis, during which a complementary
strand is built based on the sequence of a template strand.
• Differs from other sequencing technologies in that
– not rely on the optical detection of incorporated nucleotides using
fluorescence and camera scanning. This resulted in higher speed,
lower cost, and smaller instrument size.
• Ion Torrent semiconductor sequencing is performed on
either the PGM or Proton instrument depending on desired
read length and application.

7. Ion PGM & Ion proton
• Ion Torrent is the company/brand name.
• Ion Torrent's sequencers are the PGM and the Proton.
• Functionally they are very similar in that they use the same chemistry
and principles.
• The Ion Torrent PGM, was developed by Jonathan Rothberg
2010, is a small-scale, bench top instrument capable of
processing samples for
– targeted DNA and RNA sequencing, small RNA sequencing, gene
expression profiling, microbial genome sequencing, and bacterial and
viral typing applications.
• The Ion Torrent Proton sequencer is a larger-scale instrument designed
for high-throughput sequencing of exomes, transcriptomes and
genomes.

9. Fig. Semiconductor sequencing chips. The table summarizes statistics for various sequencing runs and their assessments for
each chip type: 314, 316, 318 Ion Torrent PGM, and Ion Proton (IP1/IP2/IP3). All runs were based on 200 bp chemistry and
OneTouch™ template preparation with Torrent Suite server version 2.2. Q20 refers to 99 % accuracy of a base call. 1Data
were obtained from laboratory at the University of Florida. 2Analysis includes assembly and initial annotation of a given
sequencing run using a High Performance Computer Cluster (64 Intel(R) Xeon(R) X7550 2.00GHz CPUs, 512GB of RAM, and
6TB of storage). *All Proton data were provided by Ion Torrent, Life Technologies
Andrea B. Kohn, et al. 2013, Methods Mol Biol. ;1048:247-284.

10. Principle
• Ion torrent and Ion proton
sequencing exploit the fact that
addition of a dNTP to a DNA
polymer releases an H+ ion. As each
H+ ion released will decrease the
pH. The changes in pH allow us to
determine if that base, and how
many thereof, was added to the
sequence read.

12. Procedure
• Long DNA or RNA molecules are first fragmented into a
suitable size (~50–500 nt)
• Adaptors are added and one molecule is placed onto a bead.
• Fragments, with adaptors, are PCR amplified within a water drop in
oil (emulsion PCR).
– An oil–water emulsion is created to partition small reaction vesicles that each
ideally contains one sphere, one library molecule and all the reagents needed for
amplification. Two primers that are complementary to the sequence library
adapters are present, but one is only present in solution while the other is bound
to the sphere.

16. • Each bead is placed into a single well of a slide.
• The slide is flooded with a single species of dNTP,
along with buffers and polymerase, one NTP at a
time and the process is repeated cycling through the
different dNTP species.
• The pH is detected in each of the wells.
• The dNTPs are added in a predefined flow order. At
the first release of the system, this order was a
repetitive T–A–C–G sequence.

17. Work flow

18. • The Ion torrent chip consists of a flow compartment and
solid state pH sensor micro-arrayed wells

22. Application of Ion Torrent
• At present, supported applications for Ion Torrent
semiconductor sequencing include:
– targeted DNA and RNA sequencing;
– exome sequencing;
– transcriptome sequencing;
– small RNA Sequencing;
– gene expression profiling;
– microbial or other small genome sequencing;
– bacterial and viral typing.

23. Merit and demerit of Ion torrent
• Pros.
– Semi-conductor technology, no requirement for optical
scanning and fluorescent nucleotides.
– Fast run times; a typical run takes only a few hours.
– Broad range of applications.
• Cons.
– This technology suffers from the same issue as 454 with
high error rates in homopolymers.

24. SOLiD Sequencing
• Sequencing by Oligonucleotide Ligation and Detection
• SOLiD is developed by Applied Biosystems (Life
Technologies) and commercialize since 2007.
• The sequencer adopts the technology of two-base
sequencing based on ligation sequencing.
– Ligase based sequencing

25. • Uses an adapter-ligated fragment library.
• Instead of dNTPs we add (8bp) oligo nucleotides.
• Uses an emulsion PCR approach with small
magnetic beads to amplify the fragments for
sequencing.
• Read lengths for SOLiD are user defined between
25–35 bp, and each sequencing run yields between 2–4
Gb of DNA sequence data.

26. • It is based on polony sequencing (polymerase + colony)
• The fluorescent signal recorded during the probes
complementary to the template strand and vanished by the
cleavage of probes’ last 3 bases.
• And the sequence of the fragment can be deduced after 5
round of sequencing using ladder primer sets.

27. Overview of SOLiD sequencing process
• Basically the procedure involves are:
– 1. Sample preparation
– 2. Amplification by em-PCR
– 3. Ligation reaction and imaging
– 4. Data analysis

28. Procedure
• Sample preparation
– DNA is fragmented
– Adaptors ligated to fragments
• Amplification
– Hybridization of sequence fragment
to beads
– Polonies production (polymerase
+colony) and gathering (by
centrifuge)
• Attaches to glass slide
• Ligation and reaction & imaging

30. • On a SOLiD flow cell, the libraries can be sequenced
by 8 base-probe ligation which contains
– ligation site (the first base),
– cleavage site (the fifth base), and
– 4 different fluorescent dyes (linked to the last base).

31. • A sequencing primer is hybridized to adapter
• A mixture of octamer oligonucleotides compete for ligation
to primer
• The bases in 4th and 5th position on the oligonucleotides
are encoded by one of four color labels.
• Ligated oligo is cleaved after position 5 which removes
label and the cycle is repeated.
• Whole process is repeated by off-set primer n-1
• Nucleotide read length is 30-35

33. • Fig. Applied Biosystems SOLiD sequencing by ligation. Top: SOLiD color-space coding.
Each interrogation probe is an octamer, which consists of (3 Ј -to-5 Ј direction) 2 probe-
specific bases followed by 6 degenerate bases (nnnzzz) with one of 4 fluorescent labels linked
to the 5 Ј end. The 2 probe-specific bases consist of one of 16 possible 2-base combinations.
Bottom: (A), The P1 adapter and template with annealed primer (n) is interrogated by probes
representing the 16 possible 2-base combinations. In this example, the 2 specific bases
complementary to the template are AT. (B), After annealing and ligation of the probe,
fluorescence is recorded before cleavage of the last 3 degenerate probe bases. The 5 Ј end of
the cleaved probe is phosphorylated (not shown) before the second sequencing step. (C),
Annealing and ligation of the next probe. (D), Complete extension of primer (n) through the
first round consisting of 7 cycles of ligation. (E), The product extended from primer (n) is
denatured from the adapter/template, and the second round of sequencing is performed with
primer (n Ϫ 1). With the use of progressively offset primers, in this example (n Ϫ 1), adapter
bases are sequenced, and this known sequence is used in conjunction with the color-space
coding for determining the template sequence by deconvolution. In this technology, template
bases are interrogated twice.

34. SOLiD Sequencing
5’
amplification
handle
sequencing
primersite
insert (sequence template)
1 bead loaded up with thousands of identical molecules ~ one shown for illustration
5’P
Anneal sequencing primer

35. SOLiD Sequencing
5’
AANNNIII
AG
CCNNNIII
GGNNNIII
TTNNNIII
ACNNNIII
CANNNIII
TGNNNIII
GTNNNIII
AGNNNIII
GANNNIII
TCNNNIII
ATNNNIII
TANNNIII
CGNNNIII
GCNNNIII
CTNNNIII
Sixteen probes, but only four colors
TCNNNIII

36. SOLiD Sequencing
5’
AANNNIII
AG
CCNNNIII
GGNNNIII
TTNNNIII
ACNNNIII
CANNNIII
TGNNNIII
GTNNNIII
AGNNNIII
GANNNIII
TCNNNIII
ATNNNIII
TANNNIII
CGNNNIII
GCNNNIII
CTNNNIII
TCNNNATNNN
TA CC
GGNNN

37. SOLiD Sequencing
5’
AANNNIII
AGTGCTAGATCCGAGGACTCATCGCCGATACG
CCNNNIII
GGNNNIII
TTNNNIII
ACNNNIII
CANNNIII
TGNNNIII
GTNNNIII
AGNNNIII
GANNNIII
TCNNNIII
ATNNNIII
TANNNIII
CGNNNIII
GCNNNIII
CTNNNIII
TCNNNATNNNGGNNNGTNNNTANNNGCNNN
After five cycles

38. SOLiD Sequencing
5’ AGTGCTAGATCCGAGGACTCATCGCCGATACG
CANNNTCNNNGCNNNTGNNNAGNNNCTNNN
“Reset”: Melt off previous extension products
and repeat five cycles with offset of one base

39. SOLiD Sequencing
5’ AGTGCTAGATCCGAGGACTCATCGCCGATACG
TCNNNATNNNGGNNNGTNNNTANNNGCNNN
CANNNTCNNNGCNNNTGNNNAGNNNCTNNN
ACNNNCTNNNCTNNNGANNNGCNNNTANNN
CGNNNTANNNTCNNNAGNNNCGNNNATNNN
GANNNAGNNNCCNNNGTNNNGGNNNTGNNN
...GACCCGTACGCCGTAGTGCTAGATCCGAGGACTCATCGCCGATACGAT
translate genome into color space ...
||||||||||||||||||||||||||||||
Align in color space!

40. Translating color space back to base space
2nd Base1stBase
A C G T
A
C
G
T

42. Pros and cons of SOLiD sequencing
• Pros.
– Second (after Illumina) highest throughput system
on the
market.
– The SOLiD system is widely claimed to have
lower error rates,99.94% accuracy, than most other
systems owing to the fact that each base is read
twice.
• Cons.
– Problem with palindrome sequence

44. (Buermans and Dunnen, 2014)

45. “... knowledge of sequences could contribute much to our
understanding of living matter.” [Frederick Sanger]