Biology Reference
In-Depth Information
nowadays enable researchers to sequence
complete genomes and transcriptomes in one
week. NGS machines can also be used to deter-
mine miRNA pro
blocker is chemically removed from the DNA,
thus allowing the next sequencing cycle.
With the SOLiD (Sequencing by Oligonucleo-
tide Ligation and Detection) technology, cDNA
libraries are prepared and clonal bead popula-
tions are generated in an emulsion PCR reaction
in such a way that the starting sequence of every
generated fragment is known and identical.
After enrichment for beads loaded with PCR
products, the beads are covalently deposited
onto a glass slide. Instead of a DNA polymerase
generating a second strand, the mismatch sensi-
tivity of DNA ligase is used to identify the nucle-
otide present at a given position in a DNA
molecule. This step is achieved by sequential
rounds of ligation of a mixed pool of all possible
8-mer oligonucleotides
les at an unrivaled sequence
depth. The most common high-throughput
sequencing platforms include 454 pyrosequenc-
ing (Roche), Illumina/Solexa sequencing, and
SOLiD sequencing (Applied Biosystems), which
will be introduced later in this chapter.
With the 454 pyrosequencing technology,
cDNA libraries for each sample are generated
from total RNA in the
first step. For multiplex-
ing, each cDNA library can be tagged with
speci
er sequences (barcodes) that
allow pooling of different samples for
sequencing in the same run. The cDNA frag-
ments are hybridized to small capture beads in
a water-in-oil emulsion and used for ampli
c identi
fluorescently labeled at
the 5
0
end, which also possess a cleavage site
between the
ca-
tion by PCR (emulsion PCR) so that each drop
contains a single DNA template, forming a clonal
colony in a plate containing millions of picoliter-
volume-containing wells. These wells also
contain an enzyme mix of DNA polymerase,
ATP sulfurylase, and luciferase, as pyrosequenc-
ing uses luciferase to generate a chemilumines-
cent light signal for detection of the individual
nucleotides added to the nascent DNA strand.
A camera records these signals and combines
the data to generate sequence reads. The read
lengths of up to 1 kbp, one of the advantages
of the 454 pyrosequencing technology, are not
as important when analyzing small RNAs.
Differences between NGS techniques lie in the
template immobilization strategies.
fifth and sixth nucleotide. These
molecules anneal next to the anchor sequence
to the target DNA sequence and DNA ligase
preferentially joins the molecule to the anchor
when its bases match the unknown DNA
sequence. The
first two bases are complementary
to the nucleotides being sequenced; the other
bases are degenerate and able to pair with any
nucleotides on the template sequence. When
the reaction continues, bases six to eight are
cleaved off together with the
uorescent dye.
The
fluorescence signal is informative of the
nucleotide at this position in the unknown
sequence. Multiple cycles of ligation,
uores-
cence detection, and cleavage are performed.
Following these cycles, the extension product is
removed and the template is reset with a primer
complementary to the n
e
1 position for a second,
third, and successive rounds of ligation cycles.
Therefore, each base in this sequencing method
is read twice. For more information on these
and other NGS technologies, excellent reviews
on the subject can be consulted.
71,72
The ability to multiplex allows many samples
to be analyzed in one single sequencing run,
yielding several hundred million small RNA
reads, while requiring only a few
Illumina/
Solexa use a solid-phase ampli
cation in which
one DNA molecule is attached to primers
spotted on a slide. Local clonal colonies are
then ampli
ca-
tion. The DNA can only be extended one nucle-
otide at a time; that is, four types of reversible
dye-terminator bases are used, the correct nucle-
otide is incorporated and nonincorporated
nucleotides are washed away. After a camera
has taken images of the
ed by the so-called bridge ampli
fluorescently labeled
nucleotides, the dye along with the terminal 3
0
g of total
m
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