What Is Long-Read Sequencing?

Long-read sequencing is a method that is used to determine the sequences of stretches of DNA (deoxyribonucleic acid). The cells in your body contain long strings of double-stranded DNA that are coiled up as chromosomes in a part of the cell called the nucleus, which acts as the cell’s command center. Your genes are short sections of this DNA that carry the genetic information for the growth, development, and function of your body.

In many types of living organism, including humans, the DNA exists as a two-stranded molecule, which can be thought of as being like a “ladder”, that is twisted into a shape called a double helix. Each strand is made up of units called nucleotides, which consist of a sugar molecule (deoxyribose), a phosphate group, and a nitrogenous (nitrogen-containing) base. There are four different nitrogenous bases in DNA – adenine (A), thymine (T), cytosine (C), and guanine (G) – and they bind together in pairs (A with T and G with C) to form the “rungs” of the ladder.

When new cells are made, the sequence of the nucleotides in a gene should be copied exactly. If it is not, a mutation (a change in the sequence of the DNA) results. Although some mutations have no obvious effect or can have a positive effect on the organism, enabling it to adapt better to its environment over time, others can have a significant negative effect. A number of diseases are caused by the mutation of only one nucleotide, and mutations can also lead to the development of cancer.

DNA sequencing enables the sequence of nucleotides in a piece of DNA to be determined. It is used in medicine to diagnose and treat rare diseases, identify new drug targets, and can be used as a form of genetic testing to identify if someone is at risk of developing a genetic disease and to provide counselling to affected couples who want to have a child. DNA sequencing has also helped scientists to understand the functions of genes and other parts of the genome (all of the DNA in a living organism).

How Does DNA Sequencing Work?

The first DNA sequencing technique was developed in the 1970s by Fred Sanger and his team. It involves making lots of copies of a target region of DNA in which the deoxyribose sugar molecule is replaced by a different version called dideoxyribose, in which the part of the sugar molecule that acts as a “hook” to join the strand to the next nucleotide is missing. As a result, once a dideoxyribose-containing nucleotide has been added to a DNA strand, no more nucleotides can be added and the strand ends.

The dideoxyribose-containing nucleotides are also marked with different colored fluorescent dyes, one for each of the nitrogenous bases. The different copies of the target DNA strand are then “read” in order of size and the dye color on the end of each strand is detected, which enables the sequence of the original piece of target DNA to be worked out.

Although the Sanger sequencing method can produce accurate sequences of DNA segments up to 900 nucleotides long and is still used, it is expensive and it takes a long time to sequence a large amount of DNA like a human genome. As a result, second-generation DNA sequencing techniques were developed that used “short reads” (essentially, a large number of sequencing reactions are run in parallel that sequence DNA strands that are between 50 and 70 nucleotides long), enabling large quantities of DNA to be sequenced more quickly and cheaply. Third-generation sequencing techniques are now also being developed, which include long-read sequencing, that have technical advantages over short-read sequencing.

How Does Long-Read DNA Sequencing Work?

As its name suggests, long-read sequencing can sequence long reads of DNA in one go without them needing to be broken up into smaller fragments. There are currently two companies that offer long-read sequencing and they use different methods:

1. The first involves making a copy of a long chain of DNA using the sequence of DNA that is going to be sequenced as the template, which has been joined at the ends to become circular. A single circular piece of DNA is placed on a surface with thousands of tiny little wells, so a different reaction can take place in each well. Nucleotides labelled with fluorescent dyes are then used to make new strands and the circular DNA is copied many times.
2. The second method involves a single strand of DNA being passed through a small hole, called a nanopore, in a membrane that is submerged in a salt solution. When an electrical current is established through the pore as well, each nitrogenous base blocks the flow of the current in a different way as the DNA strand passes through the nanopore. The order of these flow disruptions can then be translated into the sequence of the nitrogenous bases on the DNA strand. For both methods, many different copies of a particular sequence are then put together to form a high-accuracy “consensus” sequence.

What Is Immunohematology?

Immunohematology is a medical specialty that brings together the fields of hematology (the study of the blood and blood disorders) and immunology (the study of the immune system) in the study of antigens on red blood cells and antibodies that are associated with blood transfusions. The term “antigen” describes anything that causes a response by the immune system, while antibodies are molecules that specifically bind to antigens and identify them to the immune system as needing to be dealt with.

There are many different types of antigens on the surfaces of red blood cells. These antigens are normally ignored by the immune system, but if a person receives blood from someone else in the form of a blood transfusion, their immune system will identify and attack any red blood cells with antigens that are different to the ones on their own red blood cells. As a result, it is essential that the red blood cell antigens of both the blood donor and the recipient are determined before a transfusion is given, which enables their “blood groups” to be identified.

Red blood cell antigens are coded for by genes in our DNA called blood group systems and more than 40 have been identified in humans. Some blood group systems can have more than one form called variants. We each inherit one set of our chromosomes from our mother and another from our father, and this can mean that we inherit different variants of a blood group system. Some variants are dominant over other ones, which means that a child’s blood group may be different than the blood groups of its parents.

How Can Long-Read Sequencing Aid Immunohematology?

A number of blood group systems are very long and have complicated sequences that are difficult to work out using short-read sequencing. One of the main advantages of long-read sequencing is that it is able to span entire lengths of complicated regions of DNA. This means that it is better able to detect and sequence regions that contain a lot of repetition or for which there are several different variant forms, or variation that affects more than 50 “rungs” on a DNA “ladder”.

Long-read sequencing can also be used to work out which copy of a variant exists on which copy of a chromosome (i.e., whether it’s on the chromosome inherited from the mother or the father). New variant forms of blood group systems are still being identified, and long-read sequencing is expected to help resolve confusion about what different variant forms of these blood group systems can mean for an individual and how the genes involved are regulated. The technology may also help to establish new reference databases for all blood group systems.