Page 93 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

11.5 The Era of Whole-Genome Sequencing 387

New Techniques Sequence Millions of combination of these three innovations allows sequencing
Individual DNA Molecules in Parallel machines to record the successive addition of nucleotides to
each of millions of growing DNA molecules in real time.
The major technical advances that are making exome and Figure 11.24 outlines only one of many ingenious tech-
genome sequencing fast and cheap enough for use in iden- nologies for inexpensive high-throughput sequencing cur-
tifying disease genes permit millions of individual DNA rently under development. Other prototype systems are
molecules to be sequenced simultaneously. Many creative based on very different ideas; for example, the figure at the
methods have been invented to perform so-called high- beginning of this chapter illustrates a novel method in
throughput or massively parallel sequencing. which individual DNA molecules are threaded through
Several of these high-throughput methods for sequenc- small channels called nanopores. It is not clear which
ing human genomes are straightforward extensions of the methods will become standard in the future as costs are
Sanger sequencing by synthesis approach you already steadily driven lower. But you should have no doubt that
learned in Chapter 9, but three things are new. First, indi- the era of whole-genome sequencing has already arrived.
vidual DNA molecules being synthesized by DNA poly-
merase are anchored in one place. Second, these methods
control base addition temporally so that each base can be Disease‑Causing Mutations
identified before the next one is added. Third, in some sys- Are Hidden in a Sea of Variation
tems the sensitivity of detection is so high that a single mol-
ecule of DNA can be monitored without the need for cloning A patient’s whole-exome or whole-genome sequence
or PCR amplification steps. As shown in Fig. 11.24, the should include the sequence difference(s) responsible for

Figure 11.24 One method for high-throughput, single molecule DNA sequencing. (a) Millions of single-stranded genomic
DNA fragments, to which poly-A has been enzymatically added at the 3′ end, are hybridized to oligo-dT molecules attached to the surface of
a special microarray called a flowcell. (b) Using the genomic fragment as template and the oligo-dT as primer, DNA polymerase synthesizes
new DNA containing nucleotides with colored, base-specific fluorescent tags. These nucleotides are also blocked at their 3′ ends so that only
one nucleotide can be added at a time. (c) After a high-resolution camera photographs the fluorescence, chemicals applied to the flowcell
remove the tag and the blocking group from the just-added nucleotide. (d) Each subsequent cycle begins by infusing the flowcell with a new
dose of tagged nucleotides and polymerase, and then step (c) is iterated. The sequencing machine takes about 100 pictures that record a
sequence of colored flashes at each of the millions of spots where a single DNA molecule is being synthesized. A computer rearranges the
data into millions of short sequence reads of about 100 nucleotides and then assembles the genome sequence.
(a) Flow cell surface (c)
5' 5'
T 5' T 5'
T C 5' T C 5'
C G G C G G
C DNA fragment 1 A A C A A
A G DNA fragment 2 A A G A
G C T DNA fragment 3 C G C T
T A T T T A A T T
Oligo(dT) T A T A C T A T A G C
T A T A T A T A T A T A
T T A T A Poly-A T T A T A
T T
T A T A
T T
Add DNA polymerase + Add DNA polymerase +
labeled, blocked labeled, blocked
deoxynucleotides deoxynucleotides
(b) (d)
5' 5'
T 5' T 5'
T C 5' T C 5'
C G G C G G
C A A C A A
Blocking A G A T A G G A
group C G C T C G C T
T A A T T T A A T A T
T A T A G C T A T A G C
T A T A T A T A T A T A
T T A T A T T A T A
T T A T T A
Photograph
Photograp
T Photograph T Photographh
fluorescent
fluorescent fluorescentfluorescent
flashes flashes
Remove fluorescent
tags and blocking
groups ~100 Rounds