Sequencing instruments and their specs

• Solexa,
• Helicos,
• Roche 454, real- time
• Nano-pore sequencing
Solexa (Illumine dye sequencing)-Sequencing
by synthesis
• After the DNA is purified a DNA library, genomic library, needs to be generated. There are two ways a genomic
library can be created, sonification and tagmentation. With tagmentation, transposases randomly cuts the
DNA into sizes between 50 to 500 bp fragments and adds adaptors simultaneously. A genetic library can also
be generated by using sonification to fragment genomic DNA. Sonification fragments DNA into similar sizes
using ultrasonic sound waves.Right and left adapters will need to be attached by T7 DNA Polymerase and T4
DNA ligase after sonification.
• Adapters: Adapters contain three different segments: the sequence complementary to solid support
(oligonucleotides on flow cell), the barcode sequence (indices), and the binding site for the sequencing primer.
Indices are usually six base pairs long and are used during DNA sequence analysis to identify samples.
• Bridge amplification: Once attached, cluster generation can begin. The goal is to create hundreds of identical
strands of DNA. Some will be the forward strand; the rest, the reverse. This is why right and left adapters are
used. Clusters are generated through bridge amplification. DNA polymerase moves along a strand of DNA,
creating its complementary strand. The original strand is washed away, leaving only the reverse strand. At the
top of the reverse strand there is an adapter sequence. The DNA strand bends and attaches to the oligo that is
complementary to the top adapter sequence. Polymerases attach to the reverse strand, and its
complementary strand (which is identical to the original) is made. The now double stranded DNA is denatured
so that each strand can separately attach to an oligonucleotide sequence anchored to the flow cell. One will be
the reverse strand; the other, the forward. This process is called bridge amplification, and it happens for
Genome fragmentation
Bridge amplification
Denaturation
The sequencing occurs for millions of clusters at once, and each
cluster has ~1,000 identical copies of a DNA insert. The sequence
data is analyzed by finding fragments with overlapping areas,
called contigs, and lining them up.
Summary
Nano pore – A third generation sequencer
• USB-powered sequencers smaller than your
smartphone could revolutionize the way we
decode DNA – in hospitals, in remote locations
and even in space.
• A single molecule of DNA or RNA can be
sequenced without the need for PCR
amplification or chemical labeling of the sample.
• Do not need to make millions of extra copies of
the DNA or to label the DNA with fluorescent or
radioactive tags.
• Nano pore – a microscopic channel in the center
of a protein molecule sited in a membrane
DNA is extracted & mixed with an ionic solution. This DNA liquid is pipetted into the nanopore sequencer,
which is plugged into a laptop via a standard USB cable
Inside the nanopore sequencer, an enzyme
unwinds the DNA double helix and passes
one of the strands through nanopore.
A voltage is applied across the nanopore,
causing ions in the solution to move through
the pore and generate an ionic current. The
channel is just the right size for the DNA
strand to pass through. Since each of the
four DNA bases is different in size, it blocks a
different proportion of the nanopore as it
passes through. This means there is more or
less space for ions to pass through the
nanopore, and so more or less current can
flow across the gap. The ionic current is
measured and the data is transferred to the
computer and analyzed by specialist
software that converts the measurements in
real time into the DNA sequence – a text file
with the letters A, T, C & G.
Applications of nanopore sequencing

• Used to identify viruses, bacteria and parasites in a hospital environment.

• 2015 A team of scientists took the first sequencing field kit to West Africa and sequenced blood
samples from 142 patients during the Ebola outbreak to track the emergence of new strains.
Scientists identified the viral DNA in each sample after just 15 minutes of sequencing, showcasing
the potential of the new technology to monitor epidemics and to help identify drug and vaccine
targets to instantly inform control measures.
• In the future, nanopore sequencing could also speed up the identification of cancers, which is
particularly important for aggressive tumours; currently, it can take weeks from collecting a
patient’s DNA sample to receiving the result from a laboratory.
• Used to identify unknown species in remote areas, such as on high mountains and in the deep
sea.
• 2018 NASA astronauts had successfully identified microbes found on board the International
Space Station using nanopore DNA sequencing. This means that astronauts could potentially
diagnose infections using a sequencer to identify viral or bacterial DNA from their own mouth
swabs.
• One day, nanopore sequencers may even be used to analyse DNA on other planets.