Notes on The Advances in DNA Sequencing Technologies

Somchai Saengamnatdej, Ph.D.

December 22, 2009

The conventional DNA sequencer which is currently used in most of the

sequencing units is the one, such as automated Sanger sequencer ABI3730xl, that makes
use of the DNA synthesis in the presence of dye terminators. The followings are the
next-generation DNA sequencers.

DNA Sequencing Technologies

1. Roche/454FLX
2. Illumina/Solexa Genome Analyzer
3. Applied Biosystems SOLiD System
4. Helicos Heliscope
5. Pacific Biosciences SMRT instruments

Main Approaches
A. Pyrosequencing (i.e. in Roche/454FLX)
Principles : Pyrophosphates which are released from a DNA initiates a series of
downstream reactions that finally produce light by the firefly enzyme luciferase. The
light (varies with the number of nucleotides) is then detected.

B. Sequencing-by-synthesis approach with revesible terminators (i.e. in

Illumina/Solexa)

C. Massively parallel sequencing-by-ligation approach (i.e. in Applied Biosystems

SOLiD: the word “SOLiD” stands for “the supported oligonucleotide ligation and
detection” system)
● Each base position is effectively probed twice.
● the nucleotide is identified by analyzing the color that results from two
successive ligation reactions. (enabling the distinction between an error and
a polymorphism: an error would be detected in only one particular ligation
reaction, whereas a polymorphism would be detected in both.)

Capacities
1. Automated Sanger (ABI 3730xl) : 96 kb in 20-bp reads in a 3-hr run.
2. Roche/454FLX : 80-120 Mb in 200- to 300-bp reads in a 4-h run.

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 3. Illumina/Solexa : 1 Gb in 35-bp reads in a 2-to 3- day run.
4. ABI SOLiD : 1-3 Gb in 35-bp reads in an 8-day run.

Common Steps
1. Produce fragment libraries
● a genome is broken into blunt-ended fragments.
● anneal these fragments to platform-specific linkers.
2. Amplify single strands of a fragment library (by PCR, no cloning)
3. Perform sequencing reactions on the amplified strands
4. Image sequencing reactions in a massively parallel fashion(much higher
capacity than that of capillary sequencer, i.e. up to tens of millions of reads & 96
reads) causing longer run times.

Specific features in the amplification step

1. Helicos & Pacific:
● Also called single molecule sequencers (do not require any
amplification of DNA before sequencing)

2. Roche/454 FLX :
● PCR on agarose beads in microreactors (water-in-oil emulsion
containing PCR reactants)
● The beads' surfaces carry a type of oligonucleotides complementary
to the 454-specific adapter sequences and therefore capture a single
fragment by a specific hybridization.

3. Illumina/Solexa
● The fragments bind to the inside surface of the flow cell channels.
● Create cluster strands by bridge amplification (add unlabeled
nucleotides and enzyme polymerase, and then denature the double
stranded molecules)

4. ABI SOLiD
● Use an emulsion PCR approach with small magnetic beads

Specific features in sequencing reactions and sequence analyses

1. Roche/454 FLX
● Array the beads into a picotiter plate with several hundred thousand
wells (one bead per well)
● Add the enzyme-containing beads and mix (this enzyme will catalyze

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 pyrosequencing reaction)
● Each nucleotide flows
● A camera records the light emitted at each bead
● A software performs base-calling (error prone to homopolymer run)
● An algorithm (Newbler) assembles FLX reads.

2. Illumina/Solexa
● Add all four fluorescently labeled, 3' -OH blocked nucleotides and
DNA pol
● Remove unused chemicals after a reaction (by washing). Note that
there is one reaction because all the nucleotides are chain terminators.
● Image (laser excitation, capture the image of emitted fluorescence
from each cluster on the flow cell, record the identity of the first base
for each cluster)
● Remove the fluorescent labels and 3'-OH blocking groups to prepare
each strand for the next incorporation by DNA polymerase.
● Continue the next chemistry cycle.
● Base-calling algorithm

3. ABI SOLiD
● Deposit the magnetic beads onto a flow cell slide
● Anneal the primers to the adapter sequences on each amplified
fragment(on the beads).
● Add specific fluorescent-labeled 8 mers (at 4th &5th bases) and DNA
ligase. (These 8 mers have 16 dinucleotide combinations)
● Detect the fluorescence after each ligation step.
● Remove bases from the ligated 8mers (including the fluorescent
group)
● Prepare the extended primer for another round of ligation. (i.e.
universal primers n, n-1, n-2, n-3, and n-4 are used)

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