deoxyribose ring, the DNA polymerase is unable to form a phosphodiester bond to the phosphorus atom on the 5
0 carbon of the
next nucleotide to be incorporated. Thus, the synthesis of the DNA
chain is terminated at the point of ddNTP incorporation. When
ddNTPs that are radiolabeled or fluorescently labeled are used, the
ddNTP that incorporates on each of the various DNA strands being
synthesized can be identified. Collectively, this allows for the determination of the nucleotide at each position and thus the entire
sequence of the DNA strand.
While the initial applications of Sanger sequencing were for the
sequencing of individual genes, the method was essential for laying
the foundation of sequencing entire genomes through the technique of shotgun sequencing. Indeed, the shotgun method was
used by the Human Genome Project to determine the sequence of
the euchromatic human genome [2]. Today, however, advances in
technology have largely relegated standard chain-termination
sequencing to determining the sequence of fairly small genes. For
deciphering the genome sequence of entire organisms, Sanger
sequencing has been largely supplanted by a variety of technologies
known collectively as massively parallel or next-generation sequencing (NGS).
The two primary next-generation sequencing technology categories are short-read sequencing and long-read sequencing.
Named appropriately, the main difference between these sequencing types is the length of the individual sequencing reads produced.
Although there are various competing technologies in both the
short- and long-read sequencing markets, the short-read market
is virtually dominated by Illumina, and the long-read market is
currently split between Pacific Biosciences (PacBio) and Oxford
Nanopore Technologies (ONT).
Illumina short-read sequencing is based on the sequencing by
synthesis technique, conceptually similar to the chain-termination
technique. After fragmentation of the input DNA, a DNA library is
created by binding the small fragments to adapters. These oligonucleotides bind to the surface of the flow cell via adapters and are
then amplified using solid-phase bridge amplification, forming several million clusters of DNA. After this amplification, the sequencing occurs by a stepwise synthesis of the complementary DNA
strand using reversible-terminator dNTPs, which have a fluorophore at the 3
0 carbon of the deoxyribose ring. Like the hydroxyl
group in ddNTPs, the position of this fluorophore prevents further
elongation of the nascent chain. Laser excitation of the fluorophores allows the determination of which nucleotide was
incorporated, after which the fluorophores are chemically cleaved
off, washed away, and the 3
0 -OH group regenerated, after which
the next complementary modified nucleotide can be added [19–
21]. After the completion of several hundreds of these cycles
(depending on the selected equipment and consumables), the
Mycobacterial ID and Characterization Using WGS
405
0 carbon of the
next nucleotide to be incorporated. Thus, the synthesis of the DNA
chain is terminated at the point of ddNTP incorporation. When
ddNTPs that are radiolabeled or fluorescently labeled are used, the
ddNTP that incorporates on each of the various DNA strands being
synthesized can be identified. Collectively, this allows for the determination of the nucleotide at each position and thus the entire
sequence of the DNA strand.
While the initial applications of Sanger sequencing were for the
sequencing of individual genes, the method was essential for laying
the foundation of sequencing entire genomes through the technique of shotgun sequencing. Indeed, the shotgun method was
used by the Human Genome Project to determine the sequence of
the euchromatic human genome [2]. Today, however, advances in
technology have largely relegated standard chain-termination
sequencing to determining the sequence of fairly small genes. For
deciphering the genome sequence of entire organisms, Sanger
sequencing has been largely supplanted by a variety of technologies
known collectively as massively parallel or next-generation sequencing (NGS).
The two primary next-generation sequencing technology categories are short-read sequencing and long-read sequencing.
Named appropriately, the main difference between these sequencing types is the length of the individual sequencing reads produced.
Although there are various competing technologies in both the
short- and long-read sequencing markets, the short-read market
is virtually dominated by Illumina, and the long-read market is
currently split between Pacific Biosciences (PacBio) and Oxford
Nanopore Technologies (ONT).
Illumina short-read sequencing is based on the sequencing by
synthesis technique, conceptually similar to the chain-termination
technique. After fragmentation of the input DNA, a DNA library is
created by binding the small fragments to adapters. These oligonucleotides bind to the surface of the flow cell via adapters and are
then amplified using solid-phase bridge amplification, forming several million clusters of DNA. After this amplification, the sequencing occurs by a stepwise synthesis of the complementary DNA
strand using reversible-terminator dNTPs, which have a fluorophore at the 3
0 carbon of the deoxyribose ring. Like the hydroxyl
group in ddNTPs, the position of this fluorophore prevents further
elongation of the nascent chain. Laser excitation of the fluorophores allows the determination of which nucleotide was
incorporated, after which the fluorophores are chemically cleaved
off, washed away, and the 3
0 -OH group regenerated, after which
the next complementary modified nucleotide can be added [19–
21]. After the completion of several hundreds of these cycles
(depending on the selected equipment and consumables), the
Mycobacterial ID and Characterization Using WGS
405