VALIDATION OF NGS SEQUENCING BY SANGER SEQUENCING

VALIDATION OF SOMATIC MUTATIONS IN
CERVICAL CANCER REPORTED IN NGS
SEQUENCING BY SANGER SEQUENCING
By
N.N.P.KUMAR (KVPY FELLOW)

Under the Guidance of
Dr. Rita Mulherkar,
Principal Investigator,
Head, Dept. of Genetic Engg
Place of work :- Advanced center for
treatment, Research & Education in
Cancer (TMC)

Contents :-
• Introduction
• Cervical cancer and carcinogenesis
• NGS Sequencing
• Validation by Sanger sequencing
• Flow of work done
• Results
• Analysis
• Bibliography

Introduction :-
• Cancer of the cervix is the third most common cancer among
women worldwide after breast and colorectal cancer (Singh 2012).
• Cancer of the cervix is the most common female about 500,000
women are effected and About 300,000 women also die from the
disease annually (WHO/ICO 2010)

Genetic Alterations
• Major etiologic factor for cervical cancer
carcinogenesis is HPV, the majority of
women infected with HPV do not develop
cancer. Also, the disease generally
appears decades after initial exposure to
HPV (Ma, Wei et al. 2000).
• The genetic factors are also a major
cause of the familial aggregation in
cervical cancer, but Still not strongly
understand (Dan Chen et al. 2013)

NGS Sequencing (2011-2012 Illumina ) :-
• The behind the NGS is similar to CE
• NGS extends the process across millions of
reactions in a massively parallel fashion, rather
than being limited to a single or a few DNA
fragments
• They are capable of producing hundreds of
gigabases of data in a single run
• Types of NGS Sequencing
• Whole genome sequencing
• Exome sequencing
• Trancriptome sequencing

VALIDATION OF NGS SEQUENCING BY SANGER SEQUENCING

NGS platforms available are :
• ROCHE/454 LIFE SCIENCES
• ILLUMINA/SOLEXA
• APPLIED BIOSYSTEMS/SOLiD

Draw backs of NGS
• next-generation sequencing analysis
depend largely on the allelic fraction
as well as coverage of the mutated
residue in the tumor and normal
sample. Therefore, the NGS-based
somatic mutation detection is prone
to erroneous calls (Meyerson, Gabriel
et al. 2010; Robison 2010).
• The validation rate in these
technologies has been reported to be

Validation process work flow
• Selection of Mutation
• Primer designing
• PCR reaction
• Agarose gel electrophoresis
• DNA Purification
• Sequencing parameters adjusting
• Sanger Sequencing
• Results Validation
• Conclusion

Primer Designing
• specific primers were designed using
NCBI-Primer BLAST (
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
).
• The primer sequences flanked 200 bases
upstream and downstream to the variation
position resulting in amplicon size
between 250-400 bp.
• The obtained primers in lyophilized state
were reconstituted by adding 10mM Tris
(pH 7.5).

S.no Gene Variatio
n
Importance

1 ABCG5 aCg/aTg Involved in the regulation of dietary cholesterol absorption
(Mi-Hye Lee et.,al.2001)
2 BTNL8 gTg/gGg
Involved in immune regulation (Lucie Abeler-Dorner
et.,al.2012)
3 GRAMD1A cCc/cGc
_
4 TTN Gtc/Atc Key component in the assembly and functioning of
vertebrate striated muscles.(Gene cards data base)
S.no Gene Variation Importance

1 ABCG5 aCg/aTg Involved in the regulation of dietary cholesterol absorption
(Mi-Hye Lee et.,al.2001)
2 BTNL8 gTg/gGg
Involved in immune regulation (Lucie Abeler-Dorner et.,al.2012)
3 GRAMD1A cCc/cGc
_
4 TTN Gtc/Atc Key component in the assembly and functioning of vertebrate striated muscles.
(Gene cards data base)

DNA ISOLATION
Genomic DNA is isolated from mammalian
cells using a lysis buffer containing SDS,
EDTA and Proteinase K.
The cell and nuclear membrane lysis is
done by SDS and Proteinase K; EDTA
helps in inhibiting DNases by chelating
Mg2+ ions.

AMPLIFICATION OF GENOMIC DNA
USING PCR
• The reaction was performed in DNA Engine® Peltier
Thermal Cycler (BIO RAD). The PCR conditions used for
amplification are mentioned in the below table

Agarose Gel Electrophoresis
• Agarose gel electrophoresis is performed
to test the PCR Efficiency

PCR PURIFICATION
• After confirming amplification, the PCR
product was purified using QIAquick®
PCR Purification kit (Hilden, Germany)
as per the manufacturer’s protocol.
• Nano drop readings are taken
performed to test the Sequencing
parameters

S.no sample Concentration(ng/µl) 260/280 Ratio 260/230 Ratio
1 ABCG5(Tumor) 27.2 1.77 1.55
2 ABCG5(Blood) 22.0 1.82 1.75
3 BTNL8 (Tumor) 14.6 2.03 1.99
4 BTNL8 (Blood) 12.8 2.04 2.00
5 GRAMD1A2(Tumor) 19.2 1.91 0.88
6 GRAMD1A2 (Blood) 12.4 1.93 1.27
7 TTN (Tumor) 17.1 1.89 1.68
8 TTN (Blood) 15 2.07 1.21

Sanger Sequencing
• The Sanger method or dideoxy/chain
termination method of DNA
sequencing is gold standard for DNA
sequencing
• It is widely used for the detection of
single nucleotide variations.
• It relies on enzymatic synthesis of
DNA in vitro in the presence of chain
terminating inhibitors (the dideoxy
nucleotides or ddNTPs).

• Automation of this technique involves
labeling of each of the ddNTPs with a
different color fluorescent dye.
• These dyes fluoresce at different
wavelengths when excited by a laser.
• The resulting fluorescence is captured
by a CCD camera in the machine and
translated into an electropherogram,
which is a diagram of colored peaks
that correspond to the nucleotide in
that location in the sequence

Results and Conclusions
• All the validated somatic variations were
checked for their presence in C33A,
Caski, HeLa and SiHa cell lines by
automated Sanger sequencing.
• 5 patient samples are taken for study
• It is the first one of these type of validation

Conclusion
• 16 (out of the 27 variations selected) were
validated by Sanger sequencing (14
somatic and 2 germline).
• The validation frequency was about 60%.
• Identical mutations (as reported in
COSMIC database) in DIDO1, RNF111
and EP300 gene were validated to be
somatic in cervical cancer.

• 6 of the validated somatic variations were
present in important candidate genes of
MAPKinase pathway (FGF7, SOS2,
TGFBR2, MAP3K3, FGF22 and
RPS6KA6). Therefore, this pathway was
identified among the highly mutated ones
in case of cervical cancer.
• TTN is involved in Immune regulation

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