Sequencing and Raw Sequence Data Quality Control    ◾    7

what has been discussed above. In the library preparation step, the DNA is broken down

into fragments. The ends of these fragments are repaired and then adaptor sequences are

ligated to the ends. As Roche 454 and Ion Torrent sequencing, beads are also used and the

fragments attached to the beads are amplified using PCR. After the amplification, specific

primers are used to synthesize strands complementing the ssDNA templates and provid-

ing a free 5′ phosphate group (instead of 3′ hydroxyl group) that can ligate to one of a set

of fluorescently labeled probes. A probe is an 8-mer oligonucleotide with a fluorescent dye

at the 5′-end and a hydroxyl group at the 3′-end. The first two nucleotides of the probe spe-

cifically complement the nucleotides on the sequenced fragments. The next three nucleo-

tides are universal that can bind to any one of the four nucleotides. The remaining three

nucleotides of the probe (at the 5′-end) are also universal but are fluorescently labeled and

they are able to be cleaved during the sequencing leaving only the other five nucleotides

binding to the template strand. The set of probes consists of a combination of 16 possible

­2-nucleotides that can ligate to the primer sequence on the fragments. Every time two

specific ­nucleotides ligate, the last three nucleotides of the probe are cleaved and the fluo-

rescence specific to the ligated probe is emitted, captured, and translated into the corre-

sponding two bases. This step is followed by removing the fluorescently labeled nucleotide

and regenerating the 5′ phosphate group. The process (ligation, detection, and cleavage)

is repeated multiple times for extending the complementary strand. The strand extension

products are then removed, and a new primer is used for the second round of ligation,

detection, and cleavage. The cycles are then repeated and every time a new primer is used

for the remaining of the DNA fragments.

1.2.2.4  Illumina Technology

Illumina uses sequencing by synthesis (SBS) approach (Figure 1.4), in which it uses four

fluorescently labeled nucleotides to sequence the DNA fragments on the flow cell surface in

multiple cycles. In each cycle, a single fluorescently labeled nucleotide (dATP, dCTP, dGTP,

or dTTP) is added to the DNA growing chain. These labeled nucleotides serve as chain

terminators that stop chain extension and also the incorporation of the labeled nucleotide

in the chain causes color emission or a signal of a specific nucleotide. The signal of the

incorporated nucleotide is then captured by an imaging camera to identify that nucleotide

(or base). The terminator nucleotide is then cleaved to allow the incorporation of the next

FIGURE 1.3  Ion Torrent sequencing.