About a year after 454 machines appeared on the market, Solexa (which was
quickly purchased by Illumina) began producing NGS instruments that utilized
bridge amplification cluster generation and proprietary fluorescently tagged reversible chain terminating nucleotides. Unlike the pyrosequencing technique of 454, all
nucleotides could be run across DNA colonies at the same time. Moreover, homopolymer nucleotide runs did not pose a problem for Illumina instruments as no more
than one nucleotide could be incorporated into a chain without first unblocking the
reversible chain terminator of the incorporated nucleotide. An overview of the steps
in Illumina sequencing is provided in Fig. 5.
A third second-generation technique was developed under the name SOLiD
(sequencing by oligonucleotide ligation and detection) by Life Technologies. After
bead-based emPCR, SOLiD sequencing diverged from 454 and Illumina sequencers
in that the SOLiD system utilized extension of primers via ligation reactions using
four sets of four different fluorescently labeled di-base probes. Life Technologies
was purchased by Thermo Fisher in 2014, and consequently SOLiD sequencers are
now sold as Thermo Fisher products.
A fourth technique known as Ion Torrent sequencing was later developed by
Jonathan Rothberg, founder of 454 Technologies. Unlike the other secondgeneration techniques that detect either fluorescence or luminescence using image
capture technologies, the Ion Torrent machines detected base incorporations from
changes in pH around a bead colony (H
+ is released as a by-product of
phosphodiester bond formation). Ion Torrent instruments require no sophisticated
optics system as they do not detect fluorescence or light emission. Ion Torrent is now
owned by Thermo Fisher.
The basic operation principles of these second-generation machines are detailed
in Table 1. At present, Illumina is far and away the most widely utilized of the
technologies, a trend that does not appear to be changing anytime soon (Greenleaf
and Sidow 2014). Like 454 instruments, Ion Torrent instruments have trouble with
homopolymeric runs (Heather and Chain 2016), but their relatively low machine
cost has made them moderately popular in smaller labs. SOLiD sequencing does not
provide the read length or output of Illumina instruments, but it has a number of
unique uses, a devoted user group, and it is competitive on a cost per base basis
(Heather and Chain 2016). However, there are currently no efforts to improve
SOLiD technology (Shendure et al. 2017).
It should be noted that Complete Genomics (now owned by BGI) developed its
own sequencing platform which it has optimized for study of human genome
sequencing (Heather and Chain 2016). The Complete Genomics technology utilizes
rolling circle amplification to create “DNA nanoballs” which are fixed to a slide and
sequenced via ligation using a series of competing, fluorescently labeled probes
(Drmanac et al. 2010). Complete Genomics does not sell its instruments and, to my
knowledge, has not been involved in sequencing DNA from organisms other than
humans (www.completegenomics.com).
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D. G. Peterson and M. Arick
quickly purchased by Illumina) began producing NGS instruments that utilized
bridge amplification cluster generation and proprietary fluorescently tagged reversible chain terminating nucleotides. Unlike the pyrosequencing technique of 454, all
nucleotides could be run across DNA colonies at the same time. Moreover, homopolymer nucleotide runs did not pose a problem for Illumina instruments as no more
than one nucleotide could be incorporated into a chain without first unblocking the
reversible chain terminator of the incorporated nucleotide. An overview of the steps
in Illumina sequencing is provided in Fig. 5.
A third second-generation technique was developed under the name SOLiD
(sequencing by oligonucleotide ligation and detection) by Life Technologies. After
bead-based emPCR, SOLiD sequencing diverged from 454 and Illumina sequencers
in that the SOLiD system utilized extension of primers via ligation reactions using
four sets of four different fluorescently labeled di-base probes. Life Technologies
was purchased by Thermo Fisher in 2014, and consequently SOLiD sequencers are
now sold as Thermo Fisher products.
A fourth technique known as Ion Torrent sequencing was later developed by
Jonathan Rothberg, founder of 454 Technologies. Unlike the other secondgeneration techniques that detect either fluorescence or luminescence using image
capture technologies, the Ion Torrent machines detected base incorporations from
changes in pH around a bead colony (H
+ is released as a by-product of
phosphodiester bond formation). Ion Torrent instruments require no sophisticated
optics system as they do not detect fluorescence or light emission. Ion Torrent is now
owned by Thermo Fisher.
The basic operation principles of these second-generation machines are detailed
in Table 1. At present, Illumina is far and away the most widely utilized of the
technologies, a trend that does not appear to be changing anytime soon (Greenleaf
and Sidow 2014). Like 454 instruments, Ion Torrent instruments have trouble with
homopolymeric runs (Heather and Chain 2016), but their relatively low machine
cost has made them moderately popular in smaller labs. SOLiD sequencing does not
provide the read length or output of Illumina instruments, but it has a number of
unique uses, a devoted user group, and it is competitive on a cost per base basis
(Heather and Chain 2016). However, there are currently no efforts to improve
SOLiD technology (Shendure et al. 2017).
It should be noted that Complete Genomics (now owned by BGI) developed its
own sequencing platform which it has optimized for study of human genome
sequencing (Heather and Chain 2016). The Complete Genomics technology utilizes
rolling circle amplification to create “DNA nanoballs” which are fixed to a slide and
sequenced via ligation using a series of competing, fluorescently labeled probes
(Drmanac et al. 2010). Complete Genomics does not sell its instruments and, to my
knowledge, has not been involved in sequencing DNA from organisms other than
humans (www.completegenomics.com).
134
D. G. Peterson and M. Arick