In today’s literature, first-generation sequencing refers to automated Sanger
sequencing (and possibly the other techniques mentioned above). Though not the
first method of sequencing nor the only method of sequencing, automated Sanger
sequencing played a key role in the development of genomics as a discipline and
allowed whole eukaryote genome sequencing to be a reality. Automated Sanger
sequencing is still used today for small projects, filling in gaps in regions refractory
to NGS methods, and for BAC-end sequencing, a technique for which there is no
NGS equivalent.
3.9 Second-Generation Sequencing
Second-generation sequencing is used to describe sequencing techniques where
thousands of bead colonies/clusters are generated on, or loaded onto, a substrate.
All sequencing reactions are monitored at once. From their introduction, these
massively parallel sequencing approaches produced orders of magnitude more
DNA sequence per dollar than modern Sanger sequencing. The read lengths generated by second-generation sequencers were initially quite short compared to Sanger
sequence reads, but refinements in technology have increased second-generation
read lengths.
The first commercially available second-generation technique was developed by
454 Technologies. Characterized by emPCR followed by massively parallel
pyrosequencing of bead colonies (see Table 1 for details), 454’s successful Genome
Sequencer 20 instrument was an immediate hit leading to considerable sales and
ultimately a $60 M exclusive licensing deal with Roche. Being the first NGS on the
market gave 454 a huge advantage and led 454’s founder, Jonathan Rothberg, to say,
“It’s been a 25-year race, we're commercial, and we won” (Bio-IT World Staff
2013). However, homopolymeric runs (e.g., AAAAAAA) greater than six nucleotides in length were a problem for 454 instruments, sample preparation was relatively expensive (Metzker 2010), and Roche/454 were unable to match the
tremendous throughput increases achieved by Illumina. After sales of 454 machines
dropped precipitously, Roche halted production of 454 instruments in 2013 and
stopped sales of 454 instruments in 2015. Despite being first, 454 was outcompeted
by Illumina and Ion Torrent instruments (Hollmer 2013).
7 With this said,
454 sequencing is included in this review because it was the first commercial NGS
technology and it sparked tremendous innovation by 454 researchers and scientists
developing competing technologies. Moreover, 454 played a key role in sequencing
several plant genomes.
7 In 2012 Roche attempted a hostile takeover of Illumina which Illumina successfully thwarted.
Also, Jonathan Rothberg, the founder of 454 Technologies, later started Ion Torrent, a company
whose success effectively helped to undermine Roche’s investment in 454 (Bio-IT World Staff
2013).
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sequencing (and possibly the other techniques mentioned above). Though not the
first method of sequencing nor the only method of sequencing, automated Sanger
sequencing played a key role in the development of genomics as a discipline and
allowed whole eukaryote genome sequencing to be a reality. Automated Sanger
sequencing is still used today for small projects, filling in gaps in regions refractory
to NGS methods, and for BAC-end sequencing, a technique for which there is no
NGS equivalent.
3.9 Second-Generation Sequencing
Second-generation sequencing is used to describe sequencing techniques where
thousands of bead colonies/clusters are generated on, or loaded onto, a substrate.
All sequencing reactions are monitored at once. From their introduction, these
massively parallel sequencing approaches produced orders of magnitude more
DNA sequence per dollar than modern Sanger sequencing. The read lengths generated by second-generation sequencers were initially quite short compared to Sanger
sequence reads, but refinements in technology have increased second-generation
read lengths.
The first commercially available second-generation technique was developed by
454 Technologies. Characterized by emPCR followed by massively parallel
pyrosequencing of bead colonies (see Table 1 for details), 454’s successful Genome
Sequencer 20 instrument was an immediate hit leading to considerable sales and
ultimately a $60 M exclusive licensing deal with Roche. Being the first NGS on the
market gave 454 a huge advantage and led 454’s founder, Jonathan Rothberg, to say,
“It’s been a 25-year race, we're commercial, and we won” (Bio-IT World Staff
2013). However, homopolymeric runs (e.g., AAAAAAA) greater than six nucleotides in length were a problem for 454 instruments, sample preparation was relatively expensive (Metzker 2010), and Roche/454 were unable to match the
tremendous throughput increases achieved by Illumina. After sales of 454 machines
dropped precipitously, Roche halted production of 454 instruments in 2013 and
stopped sales of 454 instruments in 2015. Despite being first, 454 was outcompeted
by Illumina and Ion Torrent instruments (Hollmer 2013).
7 With this said,
454 sequencing is included in this review because it was the first commercial NGS
technology and it sparked tremendous innovation by 454 researchers and scientists
developing competing technologies. Moreover, 454 played a key role in sequencing
several plant genomes.
7 In 2012 Roche attempted a hostile takeover of Illumina which Illumina successfully thwarted.
Also, Jonathan Rothberg, the founder of 454 Technologies, later started Ion Torrent, a company
whose success effectively helped to undermine Roche’s investment in 454 (Bio-IT World Staff
2013).
Sequencing Plant Genomes
129