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Overview of Next-Generation Sequencing UNIT 7.31
Technologies
Barton E. Slatko,1 Andrew F. Gardner,1 and Frederick M. Ausubel2,3
1
New England Biolabs, Ipswich, Massachusetts
2
Department of Molecular Biology, Massachusetts General Hospital, Boston,
Massachusetts
3
Corresponding author: ausubel@molbio.mgh.harvard.edu

High throughput DNA sequencing methodology (next generation sequencing;

NGS) has rapidly evolved over the past 15 years and new methods are con-
tinually being commercialized. As the technology develops, so do increases in
the number of corresponding applications for basic and applied science. The
purpose of this review is to provide a compendium of NGS methodologies and
associated applications. Each brief discussion is followed by web links to the
manufacturer and/or web-based visualizations. Keyword searches, such as with
Google, may also provide helpful internet links and information.  C 2018 by

John Wiley & Sons, Inc.

Keywords: next-generation sequencing r NGS r Sanger sequencing

How to cite this article:

Slatko, B. E., Gardner, A. F., & Ausubel, F. M. (2018). Overview of
next-generation sequencing technologies. Current Protocols in Molecular
Biology, 122, e59. doi: 10.1002/cpmb.59

FOUNDING METHODOLOGY UNIT 7.4 Although relatively slow by current

The founding methods in DNA sequencing NGS standards, improvements in the Sanger
were the Sanger dideoxy synthesis (Sanger & chain termination methodology, automation,
Coulson, 1975; Sanger, Nicklen, & Coulson, and commercialization have enabled it to re-
1977; UNIT 7.4) and Maxam-Gilbert chemical main the most appropriate sequencing method
cleavage (Maxam & Gilbert, 1980; UNIT 7.5) for many current applications. Specifically, the
methods. The Maxam-Gilbert method is based replacement of ultrathin “slab gels” with mul-
on chemical modification of DNA and subse- tichannel capillary electrophoresis, the devel-
quent cleavage of the DNA backbone at sites opment of automated refillable reusable cap-
adjacent to the modified nucleotides. Sanger illaries, and “electrokinetic” sample loading,
sequencing uses specific chain-terminating have all contributed to the enhanced speed
nucleotides (dideoxy nucleotides) that lack a and ease of the Sanger process. The most
3 -OH group. Thus, no phosphodiester bond significant innovations in Sanger sequencing
can be formed by DNA polymerase, resulting have been: (1) the development of fluores-
in termination of the growing DNA chain at cent (terminator) dyes, (2) the use of thermal-
that position. The ddNTPs are radioactively cycle sequencing to reduce the quantity of
or fluorescently labeled for detection in “se- required input DNA and thermostable poly-
quencing” gels or automated sequencing ma- merases to efficiently and accurately incorpo-
chines, respectively. Although the chemistry rate the terminator dyes into the growing DNA
of the original Maxam-Gilbert method has strands, and (3) software developments to in-
been modified to help eliminate toxic reagents, terpret and analyze the sequences. The leader
the Sanger sequencing by synthesis (SBS) in automated Sanger sequencing is Applied
dideoxy method has become the sequencing Biosystems (AB; now part of ThermoFisher).
standard. The current commercialized AB sequencers
The Sanger sequencing method was devel- all utilize fluorescent dyes and capillary
oped in 1977 and is described in detail in electrophoresis (CE). The machines vary in
DNA Sequencing

Current Protocols in Molecular Biology e59, April 2018 1 of 11

Published online April 2018 in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpmb.59
Copyright C 2018 John Wiley & Sons, Inc. Supplement 122
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capacity, 4 capillaries (SeqStudio Genetic nologies in the future. We prefer to use the
Analyzer), 8 to 24 (3500 Series Genetic term second generation, third generation, etc.,
Analyzer), and 48 to 96 (3700 Series Ge- realizing that the automated AB sequencing
netic Analyzer), for DNA sequencing or machine technology described above is re-
fragment analysis protocols. All of these ally the “second” generation after the orig-
sequencers generate 600-1000 bases of accu- inal Sanger methods using radioactivity and
rate sequence. Although a variety of Sanger- slab gels. Given this naming convention, the
sequencing-based sequencing machines have need for higher throughput sequencing of
been introduced over the years, including in- large genomes at lower cost triggered the
struments from Licor, Amersham, MilliGen, development of many second-generation or
Perkin Elmer, and Dupont, all of them except “nextgen” technologies using a variety of cre-
the AB machines have been discontinued. ative methodologies in addition to automated
The Sanger sequencing technology re- Sanger sequencing. As with the commercial-
mains very useful for applications where high ization of automated Sanger sequencing, many
throughput is not required. Many DNA se- of these technologies are no longer in use (for
quencing core facilities and sequencing-for- example, SolidTM , PolinatorTM HelicosTM ).
profit companies provide Sanger sequencing These “second” generation sequencing tech-
services. The most common uses are for in- nologies and associated methods are described
dividual sequencing reactions using a specific below.
DNA primer on a specific template, for ex- Second generation sequencing methods can
ample to verify plasmid constructs or PCR be grouped into two major categories, se-
products. Now that molecular biology kits and quencing by hybridization, and sequencing by
reagents for DNA purification and relatively synthesis (SBS). SBS methods are a further de-
inexpensive high quality synthetic primers are velopment of Sanger sequencing, without the
available from many vendors, even relatively dideoxy terminators, in combination with re-
large Sanger sequencing projects can be com- peated cycles of synthesis, imaging, and meth-
pleted in a reasonable time frame and cost. ods to incorporate additional nucleotides in the
In addition to sequencing DNA, another growing chain. At first glance, these new meth-
useful application of capillary electrophore- ods may seem expensive, but the reactions are
sis on the AB machines has been the run in parallel often at nanoliter, picoliter, or
development of methods for measuring the zeptoliter volumes in small chambers, and thus
activity of selected enzymes acting upon flu- the cost per base pair sequenced is nominal.
orescently labeled DNA substrates, by anal- Continual refinements and miniaturization are
ysis, for example, of DNA fragment size reducing costs even further.
(Greenough et al., 2016). Capillary elec- A note about costs: costs for sequencing
trophoresis can also be used to simultane- encompass many variables, some of which
ously analyze multiple substrates, products are often left out of commonly presented esti-
and/or reaction intermediates in a single reac- mates of “cost per base”. The reagent costs are
tion using different fluorescent labels (Gree- usually dependent on the volume ordered and
nough et al., 2016). For example, CE was are often negotiated with the vendor. For ex-
used in high-throughput studies of DNA poly- ample, core facilities and sequencing centers
merase and DNA ligase kinetics and coupled that order in larger quantities can obtain dis-
enzyme pathways including Okazaki fragment counted pricing. Costs usually do not include
processing and ribonucleotide excision repair labor and the bioinformatics pipeline at the
(Greenough, Kelman, & Gardner, 2015; Scher- end of the process. Nevertheless, goals such as
merhorn & Gardner, 2015). AB CE is also the “$1,000 human genome” or reducing the
useful in glycobiology for analyzing fluo- “cost per base” are gold standards to be met
rescently labeled glycans (Callewaert, Gey- by the sequencing technology and research
sens, Molemans, & Contreras, 2001; Laroy, community.
Contreras, & Callewaert, 2006).
Sequencing by Hybridization
This method was originally developed
SECOND GENERATION in the 1980s, using arrayed DNA oligonu-
SEQUENCING METHODS cleotides of known sequence on filters that
The term “next generation” has implied a were hybridized to labeled fragments of the
Overview of next step in the development of DNA sequenc- DNA to be sequenced. By repeatedly hybridiz-
Next-Generation ing technology and suggests there will be a ing and washing away the unwanted non-
Sequencing
“next-next” generation naming of new tech- hybridized DNA, it was possible to determine
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whether the hybridizing labeled fragments several platforms with different technologies
matched the sequence of the DNA probes on is often used to circumvent these issues.
the filter. It was thus possible to build larger The shift from “longer read lengths” to
contiguous sequence information, based upon “short read” technologies is now trending back
overlapping information from the probe hy- toward developing technologies that generate
bridization spots. Sequencing by hybridization longer primary read lengths, while maintain-
has been largely relegated to technologies that ing the “massively parallel” nature of the tech-
depend upon using specific probes to interro- nology. This is occurring in the “third”, and
gate sequences, such as in diagnostic appli- especially the “fourth” generation technolo-
cations for identifying disease-related single- gies, described below. This is fueled in part
nucleotide polymorphisms (SNPs) in specific by cost per reaction and in part by the desire
genes or identifying gross chromosome abnor- to obtain as much primary sequence read in-
malities (rearrangements, deletions, duplica- formation as possible to circumvent sequence
tions, copy number variants or CNVs; Church, context issues such repeated DNA elements.
2006; Drmanac et al., 2002; Hanna et al., 2000; Most SBS technologies utilize a method in
Mirzabekov, 1994; Qin, Schneider, & Brenner, which the individual DNA molecules to be
2012). sequenced are distributed to millions of sep-
arate wells or chambers, or tethered to spe-
Sequencing by Synthesis (SBS) cific locations on a solid substrate. The DNA
SBS methods have taken several distinct molecules, amplified by PCR or by isothermal
approaches (Fuller et al., 2009; Mardis, 2008; modified “rolling circle” amplification meth-
Metzker, 2010; Quail et al., 2012; Shendure ods, are then subjected to DNA synthesis re-
& Ji, 2008). The “second generation” meth- actions in which labeled nucleotides, or chem-
ods generally use a solid support containing ical reactions based on the incorporation of a
micro channels or wells in which the sequenc- particular nucleotide, can be imaged or oth-
ing reactions occur. In general, most of these erwise detected. Many creative technologies
new SBS methods do not use dideoxy termi- have been developed to enable the generation
nators, although “reversible” terminators are of millions of DNA sequence reads in a single
used in some technologies, which allow the sequence run. Sequence runs may last hours
nucleotide incorporation reactions to proceed or several days depending on the throughput.
normally while imaging the incorporated nu-
cleotides, and then removing synthesis block- 454 Pyrosequencing
ing moieties on the labeled nucleotides to al- Although discontinued, we include this
low the incorporation of the next base in the as an example of the first of the “second”
sequence. generation sequencing methods that came to
Current SBS methods differ from the ap- market and utilized a novel approach, namely,
proach of the original Sanger sequencing in the detection of pyrophosphate, a byproduct
that they rely on much shorter reads (currently of nucleotide incorporation, to report whether
up to about 300 to 500 bases). Further, they a particular base was incorporated in a grow-
generally have an intrinsically higher error ing DNA chain ((Ronaghi, Karamohamed,
rate relative to Sanger sequencing, and rely Pettersson, Uhlen, & Nyren, 1996; see also
on high sequence coverage (“massively par- www.youtube.com/watch?v=WYBzbxIfuKs).
allel sequencing”) of millions to billions of Individual DNA fragments, 400 to 700 base
short DNA sequence reads (50 to 300 nu- pairs (bp) long are ligated to adapters and
cleotides) as a way to obtain an accurate se- amplified by PCR in an individual emulsion
quence based upon the identification of a con- “bead” (emPCR) reaction. DNA sequences
sensus (agreement) sequence. However, for on the beads are complementary to sequences
some technologies, sequence context errors on the adaptors, allowing the DNA fragments
occur and cannot always be corrected by in- to bind directly to the beads, ideally one
creasing the number of reads. For example, ho- fragment to each bead.
mopolymer sequences occur when DNA con- DNA synthesis followed by chemical de-
tains consecutive multiples of the same base, tection of the DNA synthesis reactions then
such as AAAAAAAAAA. In this case, se- occurs in a picoliter-sized chamber where py-
quencing platforms are limited in accurately rophosphate release is measured. By consecu-
determining the number of consecutive bases. tively flooding the chambers with sequencing
Each platform generates its own unique set reagents containing one of the 4 nucleotides,
of potential sequence context errors and users when the correct nucleotide is incorporated
DNA Sequencing
need to be aware of these limitations. Using in the synthesized strand, pyrophosphate
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release is measured utilizing a light-generating vidual well. When the sequencing reagents are
reaction. The intensity of light also provides then flowed across the wells, when the appro-
information concerning homopolymer “runs” priate nucleotide is incorporated, a hydrogen
of nucleotides in the sequence, although dif- ion is given off and the signal recorded. A ma-
ficulties are encountered with larger tracts of jor advantage of the system is that no camera,
the same nucleotide. Pyrosequencing was de- light source or scanner is needed; nucleotide
veloped in Sweden by Pyrosequencing AB, incorporation is directly converted to voltage
and subsequently acquired by Qiagen who li- which is recorded directly, greatly speeding up
censed it to 454 Life Sciences, before it was the process.
ultimately acquired by Roche. 454 sequencers The Ion Torrent system is sold by Thermo-
were discontinued in 2013, although reagents Fisher and several versions of the platform are
are still available from several suppliers. The available, including the Ion Personal Genome
“454 sequencing” technology was commonly MachineTM (PGMTM ) System, Ion ProtonTM
used for genome sequencing and metagenome System, Ion S5 system and ION S5 XL system,
samples because of the long read lengths (up to each with different throughput characteristics.
600 to 800 nt) that are typically achieved and An automated library and template prepa-
relatively high throughput (25 million bases, ration system is also available (Ion ChefTM ).
at 99% or better accuracy in a 4 hr run), facil- A large number of applications are supported,
itating genome assembly. including targeted and de novo DNA and RNA
sequencing, transcriptome sequencing, micro-
Ion Torrent (https://www.youtube.com/ bial sequencing, copy number variation de-
watch?v=WYBzbxIfuKs) tection, small RNA and miRNA sequencing,
Ion TorrentTM technology directly converts and CHIP-seq (chromatin immunoprecipita-
nucleotide sequence into digital information tion sequencing; Furey, 2012).
on a semiconductor chip (Rothberg et al.,
2011). In a DNA synthesis reaction, when a Illumina Technology
correct nucleotide is incorporated across from By far the major player in the second gen-
its complementary base in a growing DNA eration sequencing arena is Illumina, using
chain, a hydrogen ion is released. This changes technology first developed by Solexa and Lynx
the pH of the solution which can be recorded Therapeutics. Illumina sequencing is based on
as a voltage change by an ion sensor, much a technique known as “bridge amplification”
like a pH meter. If no nucleotide is incorpo- wherein DNA molecules (about 500 bp) with
rated, no voltage spike occurs. By sequentially appropriate adapters ligated on each end are
flooding and washing out a “sequencing cham- used as substrates for repeated amplification
ber” with sequencing regents which include synthesis reactions on a solid support (glass
only one of the 4 nucleotides at a time, volt- slide) that contains oligonucleotide sequences
age changes occur when the appropriate nu- complementary to a ligated adapter. (See
cleotide is incorporated. When two adjacent www.youtube.com/watch?v=womKfikWlxM).
nucleotides incorporate the same nucleotide, The oligonucleotides on the slide are spaced
two hydrogens are released and the voltage such that the DNA, following being subjected
doubles. Thus “runs” of a single nucleotide to repeated rounds of amplification, creates
can also be determined. Large homopolymer clonal “clusters” consisting of about 1000
strings of the same nucleotide are sometimes copies of each oligonucleotide fragment.
difficult to discern, however. Each glass slide can support millions of
Ion Torrent sequencing reactions occur in parallel cluster reactions. During the synthesis
millions of wells that cover a semiconductor reactions, proprietary modified nucleotides,
chip containing millions of pixels that convert corresponding to each of the four bases, each
the chemical information into sequencing in- with a different fluorescent label, are incor-
formation. To begin the process, DNA is frag- porated and then detected. The nucleotides
mented into 200 to 1500 base fragments which also act as terminators of synthesis for each
are ligated to adapters. The DNA fragments reaction, which are unblocked after detection
are attached to a bead by complementary se- for the next round of synthesis. The reactions
quences on the beads and adapters and are then are repeated for 300 or more rounds. The use
amplified on the bead by emPCR. This process of fluorescent detection increases the speed of
enables millions of beads to each have multi- detection due to direct imaging, in contrast to
Overview of ple copies of one DNA sequence. The beads camera-based imaging.
Next-Generation are then flowed across the chip containing the Illumina sequencing supports a variety
Sequencing
wells such that only one bead can enter an indi- of protocols including genomic sequencing,
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exome and targeted sequencing, metage- guides light energy into an area whose
nomics, RNA sequencing, CHIP-seq, and dimensions are small relative to the wave-
methylome methods. Different Illumina length of the illuminating light. Because of
sequencing machines provide varying levels the ZMW design and wavelength of light
of throughput, including the MiniSeq, MiSeq, utilized, imaging occurs only at the bottom
NextSeq, NovaSeq and HiSeq models. The of the ZMW where the DNA polymerase,
MiniSeq provides 7.5 Gb of information with bound to the DNA, incorporates each base
25 million reads/run in segments of 2 × 150bp in a growing chain. The four nucleotides
reads. The MiSeq can perform 2 × 300bp are labeled with different phospho-linked
reads, 25 million reads for an output of 15 Gb. fluorophores for differential detection. When
The NextSeq can provide 120Gb with 400 a nucleotide is incorporated into the growing
million reads at 2 × 150bp read length. Details chain, imaging occurs on the millisecond
on each machine and its capabilities relative to time scale as the correct fluorescently-labeled
particular sequencing projects can be found in nucleotide is bound. After incorporation,
https://www.illumina.com/systems/sequencing the phosphate-linked fluorescent moiety is
-platforms.html. released, which “floats away” from the bottom
One issue that can arise with Illumina se- of the ZMW and can no longer be detected.
quencing is a lack of synchrony in the synthesis The next nucleotide can then be incorporated.
reactions among the individual members of a Imaging is timed with the rate of nucleotide
cluster, interfering with the generation of an incorporation so that each base is identified
accurate consensus sequence and reducing the as it is incorporated into the growing DNA
number of cycles that can be performed. Care chain. This simultaneously occurs in parallel
also must be taken not to “overcluster” the in up to one million zeptoliter ZMWs, present
support, which requires accurate quantitation on a single chip within the SMRT cells.
of the amount of template DNA that is loaded Template preparation is unique in the
onto the array. Because of the large amount PacBio process as it involves production of a
of data generated, analysis of sequencing er- “SMRTbell”, a circular double-stranded DNA
rors generated from the sequencing process molecule with a known adapter sequence com-
can also be examined, aiding in the identifica- plementary to the primers used to initiate the
tion of “real” sequence variants versus proto- DNA synthesis on the template. This enables
col induced artifacts; see for example (Chen, the polymerase to read through large tem-
Liu, Evans, & Ettwiller, 2016). plates numerous times by traversing the cir-
cular molecule in each ZMW, until the poly-
merase stops, to build up a consensus sequence
“THIRD” GENERATION (LARGE (circular consensus sequence, CCS). As the
FRAGMENT SINGLE MOLECULE) adapters ligated to each side of the insert each
SEQUENCING have DNA synthesis priming sites, the se-
In contrast to second generation sequenc- quencing polymerase can traverse the circular
ing methods, third generation sequencing SMRTbell in the 5 ->3 direction on either
methods aim to sequence long DNA (and DNA strand, providing complementary infor-
RNA) molecules. The current commercialized mation from both strands of the dsSMRTbell.
technology leader in this area is Pacific An important advantage of the PacBio
Biosciences (PacBio) (https://www.youtube real time sequencing imaging and detection
.com/watch?v=v8p4ph2MAvI), which has process is that the rate of each nucleotide
commercialized two sequencing systems, addition during synthesis can be measured,
the original RSII model and more recently, termed the inter-pulse duration (IPD). Many
the SequelTM (see https://www.pacb.com/pro (but not all) nucleotides with base modifi-
ducts-and-services/pacbio-systems/). PacBio cations, such as some adenine and cytosine
sequencing, also referred to as Single methylations, change the IPD and thus can
Molecule Real Time (SMRT) sequencing, be identified as a modified base (Fang et al.,
enables very long fragments to be sequenced, 2012; Flusberg et al., 2010; Murray et al.,
up to 30 to 50 kb, or longer. The SMRT 2012; Rhoads & Au, 2015; Vilfan et al., 2013;
method involves binding an engineered Zhang, Sun, Menghe, & Zhang, 2015; see
DNA polymerase, with bound DNA to be https://s3.amazonaws.com/files.pacb.com/png
sequenced, to the bottom of a well (zero-mode /basemod_benefits_lg.png). Many different
waveguide, or ZMW, in a SMRT flow cell; see modifications can be detected and catalogued
https://www.pacb.com/smrt-science/smrt-sequ for epigenetic studies. At this point, not all
DNA Sequencing
encing/). A ZMW is a small chamber that modifications can be identified, including
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m5 C due to minor modification of the IPD. Gb of sequence could be achieved at relatively
However, chemical modification of such low cost.
nucleotides might enable their detection. Two types of nanopore systems for DNA
PacBio SMRT sequencing suffers from an sequencing are being developed, biological
inherently high error rate, but this is usually membrane systems and solid-state sensor tech-
surmounted due to the depth of the number nology. Biological nanopore sequencing relies
of read passes obtained in each ZMW for on the use of transmembrane proteins em-
each SMRTbell template. Because errors are bedded in a lipid membrane to produce the
stochastic rather than systematic, sequencing pores. Two proteins that have been utilized to
the template multiple times and aligning the generate pores have been extensively studied:
individual sequence reads results in high ac- alpha hemolysin and Mycobacterium smegma-
curacy CCS reads. Further, similar sequenc- tis porin A (MspA). The rate of DNA passage
ing of multiple templates provides additional through the pores is regulated by the addition
consensus. of motor proteins, such as a highly processive
PacBio SMRT sequencing offers several DNA polymerase (phi29) that ratchets DNA
advantages over previous methods. It enables through upon nucleotide addition. Other ac-
rapid identification of methylation sites for cessory proteins, such as a DNA helicase, ex-
epigenetic studies in addition to providing onuclease I, or oligonucleotides to bind DNA
long reads for genome assemblies. For exam- strands, enable unwinding and “ratcheting” of
ple, using PacBio technology, it is relatively the DNA nucleotides through the nanopore
straightforward to assemble a complete bacte- for detection. DNA can be moved through the
rial genome sequence using only a few SMRT pores at a constant rate for tens of thousands
cells and determine the methylation patterns of nucleotides. Solid state sensor technology
within. Often, PacBio assemblies are com- uses various metal or metal alloy substrates
bined with other methods, such as Illumina with nanometer sized pores that allow DNA or
sequencing, for increased accuracy. RNA to pass through.
In terms of throughput, the PacBio RS II Nanopore-based DNA sequencing was first
(2013) uses chips with 150,000 ZMWs, which proposed in the late 1990s and commercial-
when optimized, can generate as much as 350 ization has recently been achieved by Oxford
megabases of sequence per SMRT cell. Op- Nanopore Technologies (ONT) with a portable
timization is carried out using a Poisson dis- MinION (512 nanopore flowcell channels),
tribution so that only one polymerase bound benchtop GridION (5 mimIONs in a single
DNA molecule should be in each well. The module), and a high throughput PromethION
latest PacBio instrument, the Sequel, has 1 mil- (in development, 48 flow cells of 3000
lion ZMWs and can generate 365,000 reads, nanopores each; Greninger et al., 2015; see
with average reads of 10 to 15 kb (7.6 Gb of https://nanoporetech.com/applications/dna-na
output). Continual upgrades of the chemistry nopore-sequencing and https://nanoporetech
and technology (such as “magnetic bead load- .com/how-it-works). These sequencers use
ing”, and use of the SAGE Pippin to isolate protein nanopores in an electrically resistant
large DNA fragments for PacBio sequencing polymer membrane through which character-
(see ancillary methods below) are designed to istic current changes occur as each nucleotide
provide more, longer, and more accurate reads. passes thru the detector.
Long dsDNA molecules are first bound to a
processive enzyme, such as phi29 polymerase.
TECHNOLOGIES ON THE When the complex encounters a nanopore,
“FOURTH” GENERATION CUSP one DNA strand enters the nanopore and the
It is possible to pass long DNA molecules translocation rate through the pore is regulated
through small diameter “holes” and measure by DNA polymerase synthesis and translo-
differing currents as each nucleotide cation. The processive enzyme enables the
passes by a linked detector (Benner DNA to be continuously and processively “ra-
et al., 2007; Branton et al., 2008; Cherf cheted” through it. As a nucleotide passes
et al., 2012; Hornblower et al., 2007; through the pore, it disrupts a current that
Kasianowicz, Brandin, Branton, & Deamer, has been applied to the nanopore. Each nu-
1996; Liu, Wang, Deng, & Chen, 2016; see cleotide provides a characteristic electronic
https://www.youtube.com/watch?v=GUb1TZv signal that is recorded as a current disrup-
Overview of MWsw). In theory, more than a hundred kb of tion event. Recording is in real time and while
Next-Generation DNA could be threaded through the nanopore, 10 kb reads are now a reasonable output, in
Sequencing
and with many channels, tens to hundreds of theory 100 kb of DNA can pass through each
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nanopore and be detected. Once DNA has left bioinformatics-based image capturing/ analy-
a nanopore, the pore is available for use by a sis of fragments have led to improvements in
different DNA molecule. the technology.
Because of its small, handheld size, the Optical mapping is useful for completing
MinION has potential for many applications large genome projects (chromosome sized-
where portability and or space requirements contigs) and other applications, such as the
are at a premium. Currently the error rate is rel- identification of rearrangements in genomes. It
atively high but as with other high throughput can be used to assist in genome assembly, com-
sequencing methods, this can be circumvented pare genomic structures, and correct genome
by the large number of molecules that can assembly errors. It can also be used for com-
be sequenced. Nanopore technology has been paring strain differences, for instance in medi-
used to sequence environmental and metage- cal microbiology applications. A major advan-
nomic samples, is currently on the space sta- tage of this method is that it avoids cloning or
tion, and has been used for bacterial strain PCR artifacts and analyzes a single molecule
identification. Viral genomes, environmental at a time. Nevertheless, artifacts can be intro-
surveillance and haplotyping have been per- duced, and thus multiple fragments are utilized
formed using the MinION. Nanopore technol- to build a consensus map. The method has
ogy is also able to identify base modifications, advantages over the (previous) industry stan-
similar to PacBio technology, enabling epige- dard of pulse field gel electrophoresis (PFGE)
netic events to be readily identified. Nanopore for creating large DNA fragment chromosome
sequencing also offers direct RNA sequencing, maps. Optical mapping is less time consum-
as well as PCR-free cDNA sequencing. Thus, ing and provides, in addition to fragment siz-
nanopore sequencing has the potential to offer ing information, “roadmap” locations that can
relatively low-cost DNA and RNA sequenc- be used for building consensus chromosome
ing, environmental monitoring, and genotyp- maps (Bogas et al., 2017; Lam et al., 2012;
ing (Ammar, Paton, Torti, Shlien, & Bader, Mak et al., 2016; Mostovoy et al., 2016; Muller
2015; Cao et al., 2016; Loman, 2015; Schmidt & Westerlund, 2017; Neely, Deen, & Hofkens,
et al., 2017). 2011).
Generally, optical mapping begins by
ANCILLARY METHODS FOR shearing genomic DNA to a large size (100 mb
HIGH THROUGHPUT to 1 gb). The key is to label these large DNA
SEQUENCING AND COMPLETING fragments at specific internal sites so that the
LARGE GENOME PROJECTS labeled molecules can be imaged and lined
up to provide contiguous overlapping maps.
Optical Mapping A number of approaches can be used to label
Despite the recent advances in high specific genomic locations including digestion
throughput single molecule long-read se- of the DNA with limited single-stranded nick-
quencing methods, additional methods may ing or a rare-cutting restriction enzyme or a
be useful to complete or confirm the order set of enzymes which cleave at specific diges-
of various DNA contigs (contiguous pieces) tion sites. Labeling then can be performed on
in a genome. The most popular of these meth- the free ends with specific dyes or by enzy-
ods is “optical mapping”, using labeling meth- matic “fill–in” reactions using single-stranded
ods distributed along long DNA molecules nucleases and polymerization dye incorpora-
at nucleotide positions based upon sequence. tion. Enzymes which bind DNA at specific
These provide large scaffolds for “pinning” sites can also be used (such as DNA methyl-
known sequences onto genome maps. By cre- transferases) where a label can be transferred
ating a “visual physical map” along large to the DNA from the enzyme after binding.
DNA molecules (similar in principle to a Labeling A+T or G+C rich DNA sites can
restriction enzyme map), one can correlate also be used. The resulting fragments can be
DNA sequence with physical location. The attached to the surface of glass slides or in
method was originally developed by David C. elongated linear chambers for imaging using
Schwartz in the 1990s (Cai et al., 1995; Jing appropriate microscopy.
et al., 1998; Meng, Benson, Chada, Huff, & Once positioned on the solid substrates,
Schwartz, 1995; Schwartz et al., 1993), but the molecules are physically elongated. They
other methodologies have also been used to can then be sized and the positions of the
create the optical maps. The use of nanofludic label(s), imaged, and recorded, creating an
methods, novel ways of visualizing and identi- “optical map” of that molecule. By combining
DNA Sequencing
fying specific sites in the DNA molecules, and images from numerous DNA molecules, an
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overlapping large “consensus optical map” trifuge and the final size of the DNA frag-
can be made, which can then be used as a scaf- ments is controlled by the acceleration rate and
fold for the pinning of other physical markers, speed of the centrifuge. g-TUBE uses centrifu-
sequences, or contigs. A commercial leader in gal force (the “g” in G-TUBE) to push the sam-
this field is Bionano Genomics (San Diego) ple through a precisely manufactured orifice.
(https://bionanogenomics.com/products/). Op This produces shearing forces in the sample
Gen (https://www.opgen.com/about-us/opgen- that fragment the DNA.
overview/) will process samples as a commer- 
R
cial vendor. Diagenode, Inc. Megaruptor
The Megaruptor was designed to provide
a simple, automated, and reproducible de-
Electron Microscopy DNA Sequencing
Electron microscopy DNA sequencing is vice for the mechanical fragmentation of DNA
another single-molecule sequencing technol- from 2 kb to 75 kb. Shearing performance is
ogy, first considered in the 1960s and 70s. To independent of the source, concentration, tem-
be visualized, the DNA must be labeled with perature, or salt content of a DNA sample. The
heavy atoms as the electron microscope cannot software allows two samples to be processed
visualize individual nucleotides containing the sequentially without additional user input and
standard carbon, hydrogen, nitrogen, oxygen, without cross-contamination. Several compo-
and phosphorus, isotopes in DNA. For these nents for isolation of different sized DNA frag-
methods to work, the DNA must be denatured, ments are available. The Megaruptor base unit
labeled and stretched out on an electron mi- consists of an automated syringe pump with
croscope grid, using a “hypophase” method to attached 9-port ceramic distribution valve and
keep the DNA denatured and linear. In the- an integrated power supply. In order to control
ory, transmission electron microscopy DNA the device, a laptop with pre-loaded software
sequencing could provide extremely long read is provided.
lengths, but the issue of electron beam damage
has not been solved. A major company in this
Enzymatic Fragmentation (NEBNext
area is ZS Genetics (Wakefield MA) whose
Ultra II FS; New England Biolabs
technology involves DNA nucleotides labeled
Product Number E7805)
The NEBNext Ultra II FS enzyme in the
with three heavy atom labels: bromine, iodine
NEBNext Ultra II FS DNA Library Prep Kit
or trichloromethane. These appear as differen-
for Illumina contains the enzyme mix required
tial dark and light spots on the micrograph and
to shear a broad range of input amounts of
the fourth DNA base is unlabeled. Sequenc-
DNA into fragments for NextGen sequencing
ing by electron microscopy has not yet been
on the Illumina platform. The protocols use a
commercialized.
minimum of 100 pg and create fragment sizes
100 bp to 1 kb. The reaction is generally 5 min,
ANCILLARY TECHNOLOGIES with a 30 min heat enzymatic heat kill step at
65°C.
DNA Shearing
A key step for all DNA sequencing methods Sage Science Pippin (www.sagescience
is fragmenting DNA into a defined size. A .com/applications/dna-sequencing)
number of shearing and large fragment DNA A novel technology for generating DNA
devices are available for isolation of DNA of fragments for sequencing has been developed
various sizes for DNA library construction. by Sage Sciences. DNA from 100 bp to up
to 2 mb can be size selected and isolated
Covaris, Inc. Shearing via a modified electrophoresis device that col-
(https://covarisinc.com) lects the selected DNA size in mini-chambers.
Covaris, Inc. provides several acoustic
The method can be used for any technique
shearing devices and consumables to enable
requiring size selected DNA, including li-
fragmenting DNA and RNA to appropriate
brary construction, removing low molecular
sizes for DNA and fragment library prepa-
weight content for long-read sequencing, and
ration (150 bp to 5 kb) and also provides
preparing ultra HMW DNA libraries for long-
protocols for chromatin and RNA shearing.
range genomic applications. Several versions
For larger fragment isolation, they provide a
of their machines are available, the Pippin
G-TUBETM , designed to shear DNA into large
Prep, BluePippin, or PippinHT, in which frag-
Overview of fragments with a mean size ranging from 6 kbp
Next-Generation ments of selected lengths are collected, elimi-
to 20 kbp at room temperature (20°- 30°C).
Sequencing nating all others.
The g-TUBE works with a bench top cen-
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FUTURE OUTLOOK Callewaert, N., Geysens, S., Molemans, F., &
As DNA sequencing technology advances, Contreras, R. (2001). Ultrasensitive profiling
the goal will be faster and more accurate se- and sequencing of N-linked oligosaccharides
using standard DNA-sequencing equipment.
quencing (lower error rates, minimal artifacts) Glycobiology, 11(4), 275–281. doi: 10.1093/
with lower amounts of input DNA and RNA glycob/11.4.275.
at lower cost. Among these will be advances Cao, M. D., Ganesamoorthy, D., Elliott, A.
in sequencing from single cells and from cir- G., Zhang, H., Cooper, M. A., & Coin,
culating nucleic acids. Sequencing platforms L. J. (2016). Streaming algorithms for iden-
that are smaller, require less power (battery tification of pathogens and antibiotic resis-
operated), less reagents (zeptoliters or perhaps tance potential from real-time MinION(TM)
sequencing. Gigascience, 5(1), 32. doi:
even just a few molecules of input reagents) 10.1186/s13742-016-0137-2.
and maintenance (perhaps disposable) will be
Chen, L., Liu, P., Evans, T. C., & Ettwiller, L. M.
utilized in medical, agricultural, ecological, (2016). DNA damage is a major cause of se-
and other settings (Shendure et al., 2017). quencing errors, directly confounding variant
Higher order multiplexing (barcoding) will en- identification. bioRxiv, doi: 10.1101/070334.
able more samples to be processed in a shorter Cherf, G. M., Lieberman, K. R., Rashid, H., Lam,
time and at reduced cost. At the front end, C. E., Karplus, K., & Akeson, M. (2012). Au-
advances in robotics, liquid handling, sample tomated forward and reverse ratcheting of DNA
processing (nucleic acid preparation) will con- in a nanopore at 5-A precision. Nature Biotech-
nology, 30(4), 344–348. doi: 10.1038/nbt.2147.
tribute to these advancements. Equally impor-
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American, 294(1), 46–54. doi: 10.1038/scien-
rate bioinformatic data analysis as well as data tificamerican0106-46.
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Drmanac, R., Drmanac, S., Chui, G., Diaz, R., Hou,
A., Jin, H., . . . Little, D. (2002). Sequencing
by hybridization (SBH): Advantages, achieve-
ACKNOWLEDGEMENTS ments, and opportunities. Advances in Biochem-
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by NIH grants P30 DK040561 and P01 doi: 10.1007/3-540-45713-5_5.
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