Genome Sequencing Technology

Introduction to Sequencing

Our DNA — short for deoxyribonucleic acid — is the foundation of our identity. A single molecule contains all the genetic information necessary for our cells to replicate and maintain our body functioning properly.

In 1869, a Swiss chemist called Friedrich Miescher first identified a compound he called “nuclein” inside human white blood cells. Nearly a century later, in 1952, Rosalind Franklin was able to produce high-resolution photographs of the DNA strand.

The following year, James Watson and Francis Crick used Franklin’s photographs to discover the famous double helix structure of DNA. In 1977, Frederick Sanger invented the Sanger sequencing method — but it wasn’t until 2003 that The Human Genome Project was completed.

The Human Genome Project was a large-scale genomics project, conducted from many different countries around the world, with the aim of sequencing the entire human DNA sequence for the first time. The Human Genome Project took 13 years to complete, and it was a ground-breaking step in the field of human genetic research.

In recent years, DNA sequencing technology has become more and more accessible. As new methods have been discovered, it has become possible for anyone to have their DNA sequenced for only a few hundred dollars, which it takes only less than few days. This has opened a wide range of possibilities in many different fields, from personalised medicine to scientific research.

The Meaning of Sequencing

Base pairs are the fundamental unit of DNA. They are formed by molecules called nucleotides, which are located on opposite sides of the double helix structure, and bound together to create a “step” of the DNA ladder.

Four different nucleotides can be found in human DNA and form base pairs: adenine (A), cytosine (C), guanine (G), and thymine (T). A always binds to T, while C always binds to G as a pair. Long sequences of these pairs form DNA and chromosomes

What Is The Purpose Of Sequencing?

The order in which base pairs are arranged determines the functioning of our genes, the traits we exhibit, our ancestry and ethnic background, our metabolism, and even our risk for many diseases.

Sequencing can determine the exact order in which each of these base pairs exists in our genetic code and detect a wide range of genetic variants; the most common type of variant is called a single nucleotide polymorphism (SNP). SNPs represent a variation in a single nucleotide in a DNA strand. A human genome contains 4 to 5 million different SNPs on average.

SNPs act as biological markers that help scientists compare different DNA samples to identify both differences and similarities. When SNPs affect regulatory genes, they can impact an individual’s health or disease risk.

Sequencing can find other types of genetic variations, including:
  • Structural Variations (SVs)
  • Copy-number variation (CNV)
  • Insertion and deletions (INDELs)
  • Mitochondrial Heteroplasmy (MITO)

Decoding the human genome sequence has allowed scientists to better understand evolution, genetic inheritance, health, and many diseases. These technologies have made it possible for numerous molecular diagnostics tests, medications, and therapeutic procedures to be developed, and they’ll surely continue to contribute to scientific research for many years to come.

What Is A Sequence?

Simply put, a DNA sequence is the order in which base pairs are arranged in a section of your DNA. As we mentioned earlier, human DNA contains four different types of nucleotides: adenine (A), cytosine (C), guanine (G), and thymine (T).

DNA sequences are read in triplets — for example, ATG. Since bases are complementary and always join the same pair, it’s possible to deduce the partner triplet that corresponds to ATG on the opposite strand, which would be TAC in this example.

Genotyping vs Sequencing

If you’re interested in the field of genomics, you may have heard the terms “genotyping” and “sequencing”. While both terms refer to DNA testing, they can’t be used interchangeably since they involve very different processes.
Genotyping is a process that identifies which genetic variants are present in someone’s DNA. This method analyses specific locations in your genetic code where mutations commonly occur. A genotyping chip or microarray is often used for this purpose.
On the other hand, sequencing is used to determine the order of the base pairs that make up an entire genome. As a result, it can detect genetic variations in virtually every part of the human genome.

Why Sequencing Is Better Than Genotyping

Genotyping can only be used to identify the presence of known variants rather than new mutations. And unlike DNA sequencing methods, genotyping only reads specific portions of your DNA, looking for said variants. Genotyping uses devices called microarrays or chips.
Genotyping can still be very useful, especially if you already know which genetic variation you’re looking for. For example, someone with a family history of a certain genetic disorder could use a simple genotyping test if they only want to determine the presence of that variant.
However, it’s undeniable that sequencing can provide a lot more information than genotyping microarrays. Sequencing looks through all the base pairs in a specific DNA sequence, which allows it to identify any type of variant — whether science has already discovered its significance or not. This thoroughness means that DNA sequencing can detect much more detailed and individual genetic information.

The Use of Sequencing

A genome sequence can be obtained for many different reasons. The information gathered through this genetic testing method can be used to advance research, diagnose patients, create public health strategies, and understand infectious diseases better.
Here are some of the most common uses of sequencing DNA.
Extremely rare diseases may only affect a few hundred individuals or even just specific family groups in the entire world.

Diagnosing these diseases and identifying the mechanism that causes them can be extremely challenging. It’s also frequent for patients with rare diseases to be misdiagnosed at first, since it’s normal for physicians to initially try to rule out more common causes. But at the same time, a delayed diagnosis can lead to poor outcomes for these patients.

Genetic testing, particularly whole genome sequencing, can be extremely helpful in these cases. Since WGS reads the entire genome, it can detect any abnormalities that may be causing a rare disease. Knowing which genetic abnormality is causing a patient’s symptoms is the first step in learning more about the disease and how it can be treated.

WGS can also determine your carrier status for many genetic diseases. Even if you don’t have a disease yourself, learning this information can be very helpful for future family planning.
The quality of care a baby receives when they’re born can have long-lasting consequences on their future health and development. Newborn screening is a routine procedure that detects dozens of different diseases soon after a baby is born, even if they’re asymptomatic and healthy at the time of their birth. The conditions diagnosed by this screening panel include:

  • Amino acid disorders
  • Fatty acid oxidation disorders
  • Sickle cell disease
  • Thalassemia
  • Cystic fibrosis
  • Galactosemia
  • Congenital hypothyroidism
  • Congenital adrenal hyperplasia

Thanks to new and more affordable DNA sequencing technologies, newborn screening could supplement traditional newborn screening in the near future.

Incorporating whole genome sequencing (WGS) to newborn screening could allow doctors and parents to access a much wider range of information about the new baby. This will potentially result in a broader diagnosis, improved care, and a better quality of life for children diagnosed with a disease shortly after birth.

It’s very likely that genetic screening will become the norm in the not-so-distant future. In fact, countries such as the United Kingdom have already expressed their intention to offer routine DNA tests to their citizens.
In addition to determining your carrier status for many genetic diseases, sequencing can also assess your risk for different preventable conditions. Many illnesses are multifactorial, which means that their development depends on a combination of genetic, lifestyle, and environmental factors. Common multifactorial diseases include:

  • Heart disease
  • Diabetes
  • Hypertension
  • Alzheimer’s disease
  • Asthma
  • Thyroid disease
  • Obesity
  • Different types of cancer
  • Arthritis
  • Autoimmune disorders

Even if you can’t modify your genetic predisposition for these diseases, identifying your genetic disease risks can put you on the right track to preventing them. Through genetic testing, personalised medicine can provide customised preventive strategies that are more effective for you.

Pharmacogenomics is another branch of genetic medicine that will probably become much more popular in the near future. Through DNA sequencing, you can obtain a full pharmacogenomic profile that will tell you how you may react to specific medications and whether you’re likely to suffer adverse reactions after taking them. This will undoubtedly make it easier for physicians to prescribe treatments that fit every patient perfectly.
Not every medical diagnosis is straightforward. In some cases, a patient's symptoms can baffle an entire healthcare team. But unfortunately, a delayed diagnosis leads to delayed treatment, which can have severe repercussions for the health and life of patients.

More than 7,800 different diseases are currently known to have a Mendelian pattern of inheritance, but we only know the exact genetic cause of approximately 4,000 of those conditions. That means that, at any point in time, hundreds of thousands of people around the world could be suffering from complex diseases that can’t be easily diagnosed.

DNA sequencing can determine the exact genetic cause of a patient’s illness, thus providing a quick and accurate diagnosis for rare diseases. The incorporation of DNA sequencing into molecular diagnostics panels can make it possible for these patients to receive treatment sooner, thus improving their prognosis and quality of life.
Wanting to learn more about where we come from is a natural human urge that has always existed. Until recently, those interested in genealogy were only able to trace their family tree through painstaking research, which often led to brick walls or dead ends that couldn’t be solved.

Thanks to DNA testing, anyone can discover their ancestry from the comfort of their own home. Your DNA information can be used to determine ethnicity estimates, familial relationships, and even migratory patterns. Genetic genealogy makes it possible for brick walls to be torn down so that people can discover more extensive and thorough family trees.

Genetic genealogy is often used in combination with traditional genealogy research methods, which include:

  • Document research
  • Birth certificates
  • Marriage and divorce records
  • Death certificates
  • Newspaper articles
  • Religious records
  • Adoption records
  • Census records
  • Medical records
  • Court records
  • Oral interviews
  • Diaries or personal letters
  • Photographs
  • Tombstones

DNA tests compare the SNPs in a sample to many reference genomes in a database. Since specific SNPs can be commonly found among certain ethnic groups, identifying the SNPs in your DNA can provide ethnicity estimates.

When you upload your genetic information to different websites, you’ll also have the opportunity to be matched with other users who share some portion of your DNA so you can create your family tree.

Who Uses Sequencing?

Thanks to its wide range of applications, DNA sequencing has quickly gained popularity in many different professional fields. Anyone can benefit from discovering their DNA sequence, and below, you’ll find some of the areas in which sequencing is most useful.

Doctors and Healthcare Professionals

Physicians can use sequencing data to provide personalised treatment to their patients. As a result, next-generation sequencing technology is making it possible for patients to receive a higher quality of care than ever before. Thanks to fields such as pharmacogenomics, for example, a patient can now be prescribed a medication that will work for their condition knowing that it won’t cause side effects.

It’s also important for doctors to use genetic data to ascertain every patient’s disease risk. Having a genetic predisposition to a single, severe disease can significantly impact anyone’s well-being, and sequence data can be used to tailor preventive treatments for each patient.

Genomics also has a wide range of applications in the field of public health. Every population is different and has different needs — the DNA of a community can help public health institutions and researchers understand these needs better so they can create public health initiatives that are more effective.9 The possible benefits of genomics in the field of public health include:
  • Ability to create public health initiatives that are tailored to the needs of specific communities.
  • Enhanced understanding of infectious diseases and how to prevent them.
  • Insertion and deletions (INDELs)
  • Identification of disease risks that affect a population group.
DNA testing can also save money and strengthen healthcare systems, since diagnosing patients quickly and accurately can save resources, shorten waitlists, and reduce the workload for healthcare professionals.

How Does Sequencing Work?

Sequencing technology has come a long way since Sanger sequencing was first invented. Sequencing methods work very differently from one another, but they all usually break stretches of DNA into smaller pieces so the analysis can be performed.

First-generation sequencing

In the first-generation DNA sequencing method, such as Sanger sequencing, the molecules were first cloned into a prokaryotic plasmid and amplified within bacteria. Then, a primer was added to a denatured DNA fragment. This activates the synthesis of a single-stranded polynucleotide with the addition of an enzyme called DNA polymerase. Modified nucleotides, called dideoxynucleotides, truncated the activity of DNA polymerase, resulting in chain termination and smaller DNA fragments. The completed fragments are separated using capillary electrophoresis and analysed. Nowadays, Sanger sequencing is used to analyse shorter DNA molecules in low volumes. This method is highly accurate, despite its shortcomings. As it would be difficult to analyse large amounts of DNA bases quickly using Sanger sequencing,  modern sequencing technologies come into play.

Next-generation sequencing

Next-generation sequencing (NGS) or high-throughput sequencing methods have primarily replaced first-generation sequencing technologies, largely due to their affordability and fast turnaround times. This allows the large amounts of DNA can be analysed in parallel and deliver the results in a matter of weeks or days.

In NGS, while undertaking certain traditional steps such as DNA template generation and parallelisation, DNA samples is broken into smaller pieces, thus generating many short read sequences. Then, bioinformatics software is used to piece these sequences back together and assemble the completed read, which is later annotated with relevant findings.

Many different types of high-throughput sequencing methods have been developed, including:

  • Sequencing by synthesis (Illumina)
  • Dye sequencing (Illumina)
  • Pyrosequencing (454 Life Sciences)
  • Single-molecule real-time sequencing (Pacific Biosciences)
  • Nanopore technology sequencing
  • Sequencing by ligation (SOLiD sequencing)
  • Combinatorial probe anchor synthesis (cPAS- BGI/MGI)
  • Ion semiconductor (Ion Torrent sequencing)

Different Types of Sequencing

Different methods can be used for DNA sequencing. If you’re interested in sequencing, it’s important to understand the differences between these methods so you can choose a provider that offers exactly what you need.
Whole genome sequencing (WGS), which is also known as entire genome sequencing, is the most complete type of DNA sequencing currently available. This method is used to sequence the entirety of the human genome, including both coding and non-coding DNA. Our DNA contains approximately 3 billion base pairs, and although it may seem impossible, whole genome sequencing performs analysis on each one.

The thoroughness of this test ensures that it doesn’t miss any genetic variants, large or small, that may be skipped over by more targeted methods. Modern sequencer machines are able to complete the sequence of an entire genome faster and more accurately than ever.

All parts of your DNA are important, but they don’t play the same roles. Certain parts of your genetic sequence contain instructions for the production of many different proteins — this is called coding DNA, or exons. Your exome is the combination of all the exons in your DNA. Whole exome sequencing (WES) is a sequencing method that only determines the order of the DNA bases in your exome, rather than your whole genome. However, WES still provides plenty of information that can be useful if you want to learn more about your genetic code

Targeted exome sequencing uses a “hot-spot” or targeted sequencing panel to focus the test on specific coding regions or genes of interest. The assay uses oligonucleotide primer pairs to amplify specific regions of DNA, rather than the entire genome.

Targeted sequencing produces a greater sequencing depth that can identify low-frequency variants associated with the disease. Targeted exome sequencing is most often used in clinical settings in which the objective is simply to confirm a diagnosis, rather than screening for many different diseases and traits.

Whole exome sequencing tests obtain data on around 1% of the genome, while whole genome sequencing tests obtain data on 100% of the genome. As you may suspect, one of the main shortcomings of WES is that it doesn’t analyse important portions of non-coding DNA, which can include variants that are still relevant to many processes inside the human body.

WGS, on the other hand, is used to sequence each and every single nucleotide in the human genome. As we stated above, non-coding DNA fragments also play an important role, which is why sequencing it is just as important as sequencing the rest of your DNA.

Research also shows that WGS is more powerful than WES to detect genetic variants in the exome itself, thus making it a more accurate test.

Sequencing DNA

Interestingly, next-generation sequencing (NGS) isn’t just used for DNA sequencing. Ribonucleic acid (RNA) can also be sequenced. RNA sequencing or RNA-seq can help identify biomarkers, infer medication pathways, and assist in molecular diagnostics. Although DNA is the single molecule that contains our genetic data, single-stranded RNA forms the transcriptome of the human genome —the instructions that regulate DNA replication.

However, DNA sequencing has the most clinical applications. Now, let’s discuss some of the different methods that can be used to sequence DNA.

Short Read vs Long Read Sequencing

Short read (next generation) and long read (third-generation) sequencing refer to the length of the DNA fragments that each method can process at once. By performing analysis on longer stretches of DNA, these new methods could make the sequencing process even faster.

Next-generation sequencing methods have revolutionised DNA sequencing, but they still produce relatively short reads. Third-generation sequencing technologies, or long read sequencing, are currently being developed in the hopes of making DNA sequencing even faster and more portable.

Although long read sequencing is still prone to high error rates, it’s expected to keep improving in the near future. Soon, long read sequencing methods could become the norm and outperform existing sequencing technologies.

Genome Sequencing Cost

When sequencing technologies were first invented, they were slow and incredibly expensive. Sanger sequencing, which was the first method to be widely used for sequencing, was only able to perform short read DNA analysis. The Human Genome Project was an incredibly expensive project, costing more than $2.7 billion spread out over more than a decade of work.

But as next-generation sequencing platforms have become available to a wider audience, prices have dropped significantly. The race for the “$1,000 genome” started in 2001, with companies working tirelessly to come up with new technologies that would sequence whole human genomes for $1,000 or less.

Nowadays, it’s possible to simply order a home-based  DNA testing kit, take a sample at home, and receive your sequencing results ⁠for just a few hundred dollars.