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| There are a lot of reasons of sequencing failure, and
Macrogen normally provides resequencing service to improve sequencing
data. However, in case of sequencing problems caused by template preparation
or particularity of composition, the sequence quality will not be improved
by any conventional sequencing processes. To help customers understanding,
please refer to below for our resequencing policy and the possible reasons
for failure. |
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Resequencing is provided in order to verify any possibility of machine
error or operator's mishandling and carried out only when DNA sequencing
quality can be improved. Therefore, retrial request for
failures owing to template preparation or composition (see the below
list) will be
all charged. Also, re-sending a new batch, in spite of
the same sample names, will be regarded as a new order.
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In
case that two or more colonies
are double-picked or two or more
inserts present in the cloning
vector, multiple peaks appear
from the insert site even though
the peaks prior to the insert
may look clear (See Fig1.). |
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| Figure1. Mixed
template-plasmid Insert contamination |
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As
two or more fragments of different
lengths appear at similar electrophoretic
mobilities due to secondary structure,
multi peaks are detected after
a specific point. Sometimes these
compressed peaks may occur at
high G/C content or high A/T
content regions (See Fig2.). |
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| Figure2. Compression |
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Multiple
binding means that 2 or more peaks
with good singal strength appear
as different positions. This occurs
when more than 2 templates are mixed
or more than 2 priming sites are
present in DNA template. (Figure
3) |
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| Figure3. multiple
binding |
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Slippage
is that mixed multi peaks occur
in the downstream of long homopolymer
region. Homopolymer region is
not properly paired while polymerization,
so subsequent base slippage can
happen. (See Fig 4 -+a,b,c,d) |
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Figure4a. homopolymer
region-poly T
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Figure4b. homopolymer
region-poly A
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Figure4c. homopolymer
region-poly C
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| Figure4d. homopolymer
region-poly G |
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Within
normal sequence data, one base
may partially show double peaks.
This may be caused by SNP or
point mutation. (See Fig5.) |
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| Figure5. mixed
base |
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In
sequencing data, low peaks appear
under major peaks. This is caused
by unremoved byproduct in primer
synthesis or degradation of primer.
You may re-synthesize primers
in order to overcome this problem.
(See Fig6.) |
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| Figure6. N-1 primer |
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Similar
to the problem of N-1 primer,
in case of PCR products, minor
peaks appear under major peaks
from the specific point. |
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| Figure7. Frameshift
mutation (PCR samples) |
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After
repeat of two or more bases,
the sequencing data can become
mixed. Due to this repeat, polymerase
processing does not work well
or secondary structure may be
formed. |
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Figure8a. repeat-[GCA]
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| Figure8b. repeat-[TGC] |
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Abrupt
signal loss means that DNA sequence
signals drop rapidly. This may
be caused by strong secondary
structure of template DNA at
that point. |
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Figure9a. abrupt
signal loss- stop signal
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| Figure9b. abrupt
signal loss- drop signal |
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