High Throughput Next Generation Sequencing*

by Jad Awad
* The information in this presentation was obtained from a lecture on High Throughput NGS in an Advanced
Molecular Genetics course in King’s College London

Please refer to the slides for better visualization of the information

2 limitations of the traditional Sanger sequencing approach:

- Timing and workload: it is highly time consuming and required a lot of manual workload
and cannot be fully automated.
- Cost: the Human Genome Reference Sequence in 2003 was estimated to cost about one
billion dollars, and today, if we use a similar approach, it could cost around 10 million
dollars per sequence requiring 1 year to finish using more than 30 instruments.
Therefore, there was kind of a competition for a technological advancement of the sequencing
technologies that would then lead to the implementation of alternative solutions, particularly to
decrease the timing, workload and cost associated with traditional sequencing.
The biotech community came of with several solutions. Three main ones were:
- 454 (by Roche) approach was based on emulsion PCR and could automatically sequence
400 bp using pyrosequencing where normally the Sanger method allowed for sequencing
of 700 bps.
- SOLiD (by ABI) was also based on emulsion PCR and could sequence 35 bp through
sequencing by ligation. The drawback was that it could only read a short sequence.
- Solexa (by Illumina) approach was the winner. It used PCR on a solid support for
amplification. For sequencing, it uses the reversible terminator approach; also known as
sequencing by synthesis. The original sequence length was 35 bp (2006), but now it is
around 200 bp.

Illumina sequencing approach in a nutshell:

1- Library preparation
The genomic DNA is fragmented. The fragments are linked to adapters from each end.
2- Clonal amplification
The DNA with the adapters are attached to the surface. The adapters allow the fragments
of DNA to form bridges that will allow the starting of the amplification of the fragments.
This cycle is then repeated several times to form what is known as clusters where the
sequencing reaction will occur.
3- Sequencing by synthesis
 Each nucleotide is labeled with a different dye and the nucleotides are added along with
DNA polymerase . Only the nucleotide that is complementary to the nucleotide in the
correct position of the fragments in the clusters is added. When a complementary
nucleotide is added to a cluster, the corresponding color of light is emitted. A high tech
camera then captures it as it happens. It detects which color has been incorporated. And
just like in the Sanger method, where we had the chain terminating group on nucleotides
(ddNTPs), the Illumina sequencing method also depends on molecules to block that chain
from growing in every cycle. The clever thing about the Illumina technology is that their
reaction is reversible by adding a chemical that cleaves off the blocking group which
allows the synthesis reaction to continue. So, all of these steps of adding in the color
labeled nucleotides, imaging which color it is with a camera and then cleaving of the
blocking group is referred to as the cycle in Illumina chemistry. And that sequences a
single base for every cluster on the slide. The cycle is then repeated to sequence the
subsequent bases.
4- The images are then lined up and the sequence is determined by referring to color codes
and then the complementary to the image is what the genomic sequence is.
Ex: if we get green, red, yellow, red, yellow, then the added sequence is CTGTG and the
complementary sequence which is the one we are sequencing is GACAC.
This process happens massively in parallel with around a billion clusters on the flow cell all
which are being sequences a single base at a time.
5- After the individual sequences are determined, they are overlapped to determined the
overall sequence.
Some errors that may arise are missing nucleotides or additional nucleotides and these are dealt
with by the smart software of the machine.

Some Illumina machines can be used to do what is called RNA-seq which is short for RNA
sequencing.
This allows us to sequence the transcriptome of the cells. It involves first the isolation of RNA
using the techniques we discussed, then purify m-RNA from mixture by running on oligo-dT-
cellulose column chromatography. Then synthesize cDNA from mRNA and do then repeat the
same procedure as before.
There is also a technique called single cell RNA sequencing or scRNA-seq. This allows us to
determine the expression profiles of single cells that are isolated under a microscope. The cell is
then lysed. The mRNA is isolated, amplified using PCR and then sequenced using Illumina
machines.