Secuenciacin por Ligacin

Es un mtodo enzimtico de secuenciacin que

emplea una DNA ligasa en lugar de una
polimerasa para identificar la secuencia
objetivo.
Mtodo SOLiD.

Metodologia:
Library Preparation
1.Prepare one of the two types of libraries
(Figure 1) for SOLiD System sequencingfragment or mate-paired.

 Emulsion PCR/Bead Enrichment

2.Prepare clonal bead populations (Figure 2).
3.After PCR, denature the templates and
perform bead enrichment to separate beads
with extended templates from undesired
beads.

 Bead Deposition
4.Deposit 3 modified beads onto a glass slide
(Figure 3). During bead loading, deposition
chambers enable you to segment a slide into
one, four, or eight sections.

Sequencing by Ligation
5.Primers hybridize to the
P1 adapter sequence on
the templated beads
(Figure 4).
6.A set of four
fluorescently labeled dibase probes compete for
ligation to the sequencing
primer.

7.Multiple cycles of
ligation, with the number
of cycles determining the
eventual read length.
8.Following a series of
ligation cycles, the
extension product is
removed and the
template is reset with a
primer complementary to
the n-1 position for a
second round of ligation
cycles.

 Primer Reset
9.Five rounds of primer reset are completed
for each sequence tag (Figure 5). Through the
primer reset process, virtually every base is
interrogated in two independent ligation
reactions by two different primers.

 Exact Call Chemistry

10.Up to 99.99% accuracy is achieved with the
Exact Call Chemistry Module by sequencing
with an additional primer using a multi-base
encoding scheme.

Secuenciacin por Sntesis

Esta tcnica fue desarrollada en la Universidad
de Cambridge por Shankar Balasubramanian
and David Klenerman.
Se basa en un mtodo similar al SANGER .

 Los fragmentos a secuenciar son preparados con

adaptadores en los extremos, para permitir que
sus cadenas desnaturalizadas puedan unirse a
una superficie slida y as poder ser enriquecidas
por medio de una amplificacin tipo puente,
generando clster de cadenas del mismo
fragmento, con el fin de amplificar la seal
generada por los nucletidos fluorescentes
incorporados a la cadena en crecimiento y as
puedan ser detectados por una cmara, esto
durante un proceso cclico que permite alargar las
lecturas dependiendo de la plataforma
seleccionada.

Referencias:
http://langebio.cinvestav.mx/labsergen/
http://nxseq.bitesizebio.com/articles/sequencing-bysynthesis-explaining-the-illumina-sequencing-technology/
http://www.appliedbiosystems.com/absite/us/en/home/ap
plications-technologies/solid-next-generationsequencing/next-generation-systems/solid-sequencingchemistry.html?sa=t&rct=j&q=&esrc=s&source=web&cd=9
&ved=0CF0QFjAI&url=http%3A%2F%2Fwww3.appliedbiosy
stems.com%2FAB_Home%2Fapplicationstechnologies%2FS
OLiDSystemSequencing%2FOverviewofSOLiDSequencingCh
emistry%2F&ei=L5BRUo64K4vS9QTj4C4DQ&usg=AFQjCNEp3FL_otaMG4Yp86AVwynEip8PIQ&s
ig2=L1ZmPCvJDPBXrftYiZyzJQ&bvm=bv.53537100,d.eWU