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Principles and Workflow of Whole Exome

Sequencing
As the development of biological experimental technology, especially gene-sequencing technology,
both laboratory and clinic researchers realize that genome sequencing is the best way to analyze the
etiology, pathophysiology, treatment and prognosis of diseases. Researches further demonstrate that
there are only 30 million base pairs of genes that contain essential information of proteins for human
beings.

The exome is ususally defined as the sequence encompassing all exons of protein coding genes, as
well as nonprotein coding elements such as microRNA or lncRNA. The investigation of exome helps to
figure out which loci are responsible for proper diseases. When researchers plan to explore exons
information of human genome, the cost to whole genome sequencing will be quite surprising
considering the total length of human genome is over 3 billion base pairs in size. To study rare
mendelian diseases, exome sequencing is a more effective way to identify the genetic variants. The
breakthrough of target-enrichment strategies and DNA sequencing techniques contributes to the
development of whole exome sequencing.

Principle of exome sequencing

Exome sequencing contains two main processes, namely target-enrichment and sequencing.
Target-enrichment is to select and capture exome from DNA samples. There are two major methods to
achieve the enrichment of exome.

⚫ Array-based exome enrichment uses probes bound to high-density microarrays to capture

exome. A microarray is a 2 dimensional array on glass slide or silicon thin-film, which contains
oligonucleotides complementary to target genome parts. While the fragmented DNA samples flow
through microarray, the complementary pairing effect will force exome binding at microarray, with
the other parts of genome remain dissociative, which results in the separation of exome from other
parts of genome.

⚫ In-solute capturing is based on magnetic bead. Magnetic bead is a kind of magnetic nanoparticles
which contain functional chemical components to combine target substances. In this case,
magnetic beads which could bind exome are used. Then the story is just the same with array-based
method, exome is attracted and bound to the magnetic beads, with other parts of genome remain
dissociative. The advantage of in-solute capturing method is that the usage of magnetic bead
allows the reaction to be more effective by shaking or heating the system.

Both of the methods are effective ways to extract exome from genome. So we say the sensitivities of
both are high enough. However, the problem is specificity. There are parts of genome which share the
similar sequence of some exons. Those parts of genome may bind to microarray or magnetic beads,
resulting in false positive.

Sequencing is the process to figure out the arrangement of all the deoxyribonucleotides in exome,
which may help us to understand the potential pathophysiology alternation in some diseases. Because
of the decrease of the cost, the importance of whole exome sequencing is prominent. The cost of
human genome is approximately equal to two or three times the cost of whole exome sequencing. So
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why not run more samples using whole exome sequencing to obtain more statistically significant result?

General workflow of exome sequencing

Here a common workflow of exome sequencing is shown as below. The instructions of the major
processes in the workflow will be discussed below.

Figure1. Workflow of whole exome sequencing. Notice that the detailed procedures are various from different
types of samples, reagent kits and sequencing instruments. Researchers should follow the instructions of reagent,
kits and sequencing instruments.

⚫ Prepare your DNA samples: DNA fragmentation

Almost all the experiment on DNA begins with DNA fragmentation. DNA should be sheared into proper
pieces, because usually the length of DNA sample extracted from tissues or cells is too long. This
shearing process is called DNA fragmentation. Effective target length is determined by the sequencing
instrument that you choose. In order to process whole exome sequencing, there are several major ways
to fragmentize DNA samples.

 Physical fragmentation. Physical fragmentation includes acoustic shearing, sonication and

hydrodynamic shear. Among them acoustic shearing and sonication are the main methods for DNA
fragmentation. DNA samples are broken into several pieces due to the acoustic cavitation and
hydrodynamic shearing when they are exposed to ultrasound.
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 Enzymatic Methods. Enzymes used to break DNA into small pieces include nuclease or
transposase. Nuclease will cleave the phosphodiester bonds between nucleic acids, resulting in the
breaking down of DNA. Specifically, restriction endonuclease will cleave DNA at restriction sites.
Transposase is used to mediate transposition events, processes that a certain DNA segment could
"move around" the chromosome. It also plays a role in DNA fragmentation if we prepare appropriate
DNA samples and transposase. The fragmentized DNA is linked with adapters instead of inserting
again, resulting in fragmentation.

After fragmentation, your DNA samples are ready for target-enrichment process.

⚫ Isolation of exome: target-enrichment methods

Exome has to be isolated from human genome before sequencing as the former contributes to only 1%
of the latter. The process of capturing the target genomic regions is called target-enrichment. The basic
idea of target-enrichment is to separate anything of interest from other substances using the
physicochemical property difference between them. There are some common kits of target-enrichment
methods. No matter what kit you choose, the variability in capture influence your exome sequencing, so
be aware to the quality, quantity and fragment sizes of your DNA samples.

Table1. Common kits of target-enrichment for sequencing.

Kits Targeted Genomic DNA Adapter Probe Length

Region Input Required Addition (mer)
Agilent SureSelect XT2 V6 Exome 60 Mb 100 ng Ligation 120
Agilent SureSelect XT2 V5 Exome 51 Mb 100 ng Ligation 120
IDT xGEN Exome Panel 39 Mb 500 ng Ligation not described
Illumina Nextera Rapid Capture Expanded 62 Mb 50 ng Transposase 95
Exome
Roche NimblegenSeqCap EZ Exome v3.0 64 Mb 1 ug Ligation 60 - 90

⚫ Harvest your products: washing and elution

After the separation of exome and other parts of genome, several times of washing are required. The
process of washing is just like what this word means literally -- to wash out anything we do not want so
as to keep the thing of interest. In this case, we do not want substances such as the other parts of
genome, proteins, and electrolytes. Distilled water is usually used to elute target, but some special
reagent kits may require specific eluent. Eluent is the reagent to wash down the exome from microarray
or magnetic beads, which is able to break the connection between exome and binding substances. Both
washing and elution process could be processed multiple times in order to obtain purer exomes. Also in
some cases, one more target-enrichment process is performed to make the elution better. Just follow
the instruction of reagent kit you used, and adjust your protocol according to your actual situation.

⚫ Sequencing technology

Because of time cost and length requirement of the Sanger Sequencing, the sequencing technology did
not contribute much in biological and clinical studies, until next generation sequencing (NGS)
technologies are invented. NGS technologies are based on the usage of dyed ddNTPs in Sanger
method. The improvement is that NGS allows DNA strands to be combined, amplified and detected at
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the same time, leading to breakneck increase in length requirement and efficiency of sequencing. To
simplify, the principle of NGS is to bind the exome samples in a proper base (such as flowcell of Illumina
Hiseq and magnetic beads of Roche-454) and replicate them by PCR-in-situ, in order to make signal in
every rounds of elongation amplified. Then ddNTPs are detected after every round of elongation. Finally,
the complete sequence is integrated using biological information algorithm. NGS largely improves the
efficiency and allows higher-throughput detection, that is why NGS is also called high-throughput
sequencing and is widely used.

Besides of NGS, the third generation of sequencing is developing rapidly, which largely exceeds the
efficiency of NGS. The key feature of third generation sequencing is single-molecule sequencing. It
shortens the time cost of whole genome sequencing to several minutes. Companies such as PacificBio
and Oxford Nanopore have proved their method works, and third generation of sequencing technology
could lead a revolution in exome sequencing area.

Here are some common methods of whole exome sequencing used nowadays.

Table2. Common methods used for sequencing nowadays.

Methods Company Generation Read Accuracy Reads per run Time per run
length
Ion Ion Torrent 2nd Up to 600 99.60% up to 80 million 2 hours
semiconductor generation bp
Pyrosequencing Roche 2nd 700 bp 99.90% 1 million 24 hours
(454) generation
Sequencing by Illumina 2nd 75-300 bp 99.90% 1 million to 3 1 to 11 days
synthesis generation billion
Sequencing by ABI 2nd 50+35 or 99.90% 1.2 to 1.4 billion 1 to 2 weeks
ligation (SOLiD) generation 50+50 bp
Nanopore Oxford 3rd up to 500 92–97% dependent on 1 min to 48
Sequencing Nanopore generation kb (single read length hours
Technologies read)* selected by
user
Single-molecule Pacific 3rd 30,000 bp 87% 10-20 billion 0.5-20 hours
real-time Biosciences generation (single
sequencing read)*
*For third generation sequencing, accuracy is usually improved by sequencing for multiple times.

⚫ Data analysis

The data of sequencing are confusing and unreadable before bioinformatics analysis and interpretation,
because most of the sequencing methods produce short fragments of sequence, which require
sequence assembly to figure out the final result. The following pipeline can be used by researchers who
are interested in performing WES analysis for variant calling and genetic diseases.
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Figure2. The typical variant calling pipeline.

Conclusion

We have benefited a lot from exome sequencing in both academic research and clinic diagnosis.
Thanks to exome sequencing, the understanding of genome is developed to a new level. Many
diseases used to be mysteries, such as neurological disorder in infants, which could be predicted now.
Furthermore, many diseases with few treatments, such as carcinoma, are allowed to be treated by
targeted therapy. It is said that the fourth generation of sequencing technology is developing. Hope it
would drive another revolution in biological and medical research.
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