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Pyrosequencing:
1. non-electrophoretic,
2. bioluminescence method that measures the
release of inorganic pyrophosphate by
proportionally converting it into visible light
using a series of enzymatic reaction
3. Nucleotide incorporation generates light seen as
a peak in the Pyrogram trace
Applications
1. Whole genome sequencing
2. Targeted resequencing
3. Sequencing-based
4. Transcriptome Analysis
5. Metagenomics
isothermal amplification instead of emulsion
Illumina/Solexa 1. Solid-phase amplification
2. can produce 100-200 million spatially separated
1. Most popular sequencing clusters, providing free ends to which a
technique nowadays! universal sequencing primer can be hybridized
2. , 100-150bp reads are used. to initiate the NGS reaction
3. longer fragments are ligated to 3. Reversible terminator sequencing method is used by
generic adaptors and annealed to a Illumina/ in which modified nucleotides are
slide using the adaptors. used in reversible termination.
4. PCR Amplification by Bridge 4. Reversible termination uses bridge PCR,
Amplification improving the efficiency of this stage of the
5. This technique was pioneered by process.
Illumina, with their HiSeq and a. Reversible terminators can be grouped
MiSeq platforms. into two categories:
a. HiSeq is the cheapest of i. 3-O-blocked reversible
the second generation terminators and
sequencers with a cost of ii. 3-unblocked reversible
$0.02 per million bases terminators.
b. . It also has a high data
output of 600 Gb per run
which takes around 8 days
to complete.
Sequencing by Cyclic Reversible Termination
(CRT):
CRT uses reversible terminators in a cyclic
method that comprises
1. nucleotide incorporation,
2. fluorescence imaging and
3. cleavage
nucleotides that have not been 4. DNA polymerase, bound to the primed
incorporated are washed away and the
template, adds or incorporates just one
fluorescent branch is cleaved using TCEP
fluorescently modified nucleotide
(tris(2-carboxyethyl)phosphine). TCEP also
removes the 3-O-azidomethyl group,
5. Unincorporated nucleotides are washed
regenerating 3-OH, and the cycle can be
away and a four-color imaging is
repeated
acquired by total internal reflection
fluorescence (TIFR) using two laser 3