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CHAPTER 11 fourth, radiolabeled dNTP. Each reaction consisted of the DNA tem-
plate to be sequenced in a mixture containing a DNA primer, a DNA
GENOMICS polymerase, a mixture of four dNTPs, and one of the four ddNTPs.
Here, the chemistry of ddNTPs, which lack the 3′ hydroxyl group pres-
ent in a native dNTP, resulted in chain termination when incorporated
into a growing DNA chain, as DNA polymerase cannot add another
Lukas D. Wartman and Elaine R. Mardis nucleoside without the 3′ hydroxyl group present. With multiple rounds
of primer elongation, the ddNTPs incorporate randomly in the newly
synthesized strands according to the complementary nucleotides of the
SUMMARY DNA template. By denaturing the newly synthesized strands from the
DNA templates and resolving each of the four DNA fragment mixtures
The introduction of next-generation sequencing platforms, coincident with on separate lanes by gel electrophoresis, one could read out the sequence
genome-scale preparatory and analytical approaches and the completion of of the DNA template from the resulting autoradiograph. Significant
improvements to the original Sanger sequencing protocol included the
the Human Genome Reference, has ushered in the era of genomics. This chap- use of fluorescently labeled ddNTPs to allow for sequencing to occur in
ter introduces the fundamentals of next-generation sequencing methods, one reaction rather than four, improved thermally stable DNA poly-
3
provides an overview of the basics of data analysis, and explores the myriad merases that permitted temperature cycling (“cycled sequencing”), and
applications developed to exploit the scale and throughput of next-generation the use of capillary electrophoresis rather than standard gel electropho-
sequencing toward questions of biomedical importance. Specifics of cancer resis for automated separation matrix filling between samples. Mod-
4–7
genomics, complex disease genomics, and how they pertain to hematologic ern Sanger capillary sequencers typically generate DNA sequencing
basic science and clinical practice are discussed, along with the modern-day reads in the range of 400 to 900 base pairs (bp). The main limitation of
realities of the consenting process. Sanger sequencing is that the sequencing reaction is decoupled from
the electrophoretic separation and detection steps. To piece together the
sequence for a large segment of DNA or entire genome, genomic DNA
must be randomly fragmented and subcloned into a bacterial vector,
with each cloned DNA isolated and sequenced. The resulting sequenc-
HISTORY OF GENOMICS: SANGER ing reads are assembled computationally to recreate larger fragments
SEQUENCING that recapitulate the starting DNA nucleotide sequence. This process is
expensive, time-consuming, and laborious. However, with the availabil-
The scientific discipline known as genomics has dramatically changed ity of robotic DNA isolation and sequencing reactions, coupled with
since the publication of the Human Reference Genome in 2003, primar- high-throughput capillary sequencers, the human genome, among the
ily as a result of the introduction and broad-based implementation of genomes of many other organisms, was decoded. Currently, Sanger
new sequencing technologies. Prior to the mid-2000s, Sanger sequenc- sequencing is still in use to complete smaller scale sequencing projects
1
ing was the predominant DNA sequencing approach, and was used to and to validate findings from next-generation sequencing studies.
complete the sequencing of the first human reference genome. Freder-
ick Sanger and his colleagues developed Sanger or “chain termination”
sequencing in the late 1970s. In their original method, four reactions MODERN GENOMICS:
2
were used to accomplish chain termination by incorporating separate
di-deoxynucleoside triphosphates (ddNTPs), each included with a mix NEXT-GENERATION SEQUENCING
of three unmodified deoxynucleoside triphosphates (dNTPs) and a
OVERVIEW OF NEXT-GENERATION
SEQUENCING

The method for next-generation sequencing (NGS), or massively par-
Acronyms and Abbreviations AML, acute myeloid leukemia; ATAC-seq, uses the allel digital sequencing, is distinct from Sanger sequencing in that the
hyperactive Tn5 transposase to simultaneously fragment and add sequencing adap- sequencing reactions alternate with cycles of signal detection to provide
tors to accessible DNA; bp, base pair; ChIP-seq, chromatin immunoprecipitation the data readout at a significantly accelerated scale. The use of NGS
8,9
sequencing; ddNTP, di-deoxynucleotide triphosphate; DNase-seq, uses DNase I to in the years after the completion of the Human Genome Project has
fragment DNA based on DNase I hypersensitive sites as a marker of chromatin acces- greatly increased the use of genomics and has significantly impacted
sibility; dNTP, deoxynucleotide triphosphate; FAIRE-seq, formalin crosslinking of DNA the pace of biomedical research. Although there are several different
10
to proteins prior to random fragmentation; FFPE, formalin-fixed, paraffin embedded; NGS platforms offered commercially, they are methodologically quite
FLT3-ITD, internal tandem duplications of FLT3 gene; Gb, gigabase, i.e., billion base similar. Unlike Sanger sequencing, NGS does not require subcloning of
pairs; GINA, The Genetic Information Nondiscrimination Act; GWAS, genome-wide DNA, propagation in a bacterial host, and isolation of individual tem-
association study; lncRNA, long noncoding RNA; indel, term for the insertion or the plates prior to sequencing. Instead, DNA is randomly fragmented into
deletion of bases; MDS, myelodysplastic syndromes; miRNA, microRNA; MNase-seq, a pool of small pieces (generally 100 to 500 bp) and then ligated with
micrococcal nuclease (MNase) determines nucleosomal footprints and boundaries specific synthetic DNA linkers (or adaptors) at the fragment ends to
by pairing with NGS as a marker of chromatin accessibility; MRD, minimal residual generate a NGS “library.” The library fragments are subsequently ampli-
disease; NGS, next-generation sequencing; PCR, polymerase chain reaction; RNA- fied by a process that isolates individual library fragments to a specific
seq, RNA sequencing; siRNA, short-interfering RNA; SNP, single nucleotide polymor- location prior to amplification. In general, this in situ amplification
phism; snoRNA, small nucleolar RNA; snRNA, small nuclear RNA; Tb, terabase, i.e., occurs on a covalently modified surface (a bead or flat silicon surface)
trillion base pairs; WGBS, whole-genome bisulfite sequencing; ZMW, zero-mode with complementary linkers covalently attached to it, using a specific
waveguide. dilution of library fragments as input. In this step, the individual library
fragment amplification permits sufficient signal output for detection

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