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White Paper
INTRODUCTION: Human genome sequencing project completion is considered as a
major milestone in genomic research. This global effort had established a new vision of
advance therapeutics. It was mainly accomplished by conventional ‘Sanger Sequencing’
method, which had taken rigorous efforts of lab workers for 12 years and approximately $3
billion were spent. On similar lines, Prof. Craig Venter‘s lab also sequenced a human genome.
However, successful completion of these two competing human genome projects could not
satisfy biologists’ hunger for greater sequencing throughput and more economical sequencing.
The main limitations of Sanger method were high cost, low speed and labour intensive nature.
Due to these limitations, the Sanger sequencing process could not fulfill the rapid increase in
demand from pharma industries and genomic research facilities. This vacuum was successfully
filled by next generation sequencing methods, as they are cost effective, less time consuming
and capable enough to read millions of DNA sequences in parallel.
NGS methods
are cost effective,
less time consuming
and capable enough
to read millions of
DNA sequences in
parallel.
“
“
Next generation
sequencing-
advancement in
Pharmaceutical
Research
White Paper
According to recent facts from National Human Genome Research Institute, an estimated cost for the human genome
sequencing project is ~$4211. This signifies a greater reduction in cost when compared with first human genome sequencing.
Credit, for this, goes to advancement in sequencing methodologies i.e. parallel reading of DNA sequences.
Considering the reading depth and large coverage capabilities of NGS methods a more ambitious `1000 genome sequencing
project’ has been accomplished. The main aim of this project is to find all possible mutations in human genome, which might
be responsible for a disease condition. This data repository of 1000 genome project is growing rapidly. In fact, a recent
report indicates accomplishment of 2504 human genomes for the same. Clearly, the growth of genomic data in the biological
repository is in agreement with the advancement of NGS technology.
NGS IN PHARMA RESEARCH: Non-Sanger based sequencing technologies provided
a tremendous boost to the scientific community, thereby enabling them to
discover novel facts and achievements. This changed their way of thinking, thus providing
deeper insights into our genetic makeup. However, the significant contribution of Sanger
sequencing had created a comfort zone within the scientific fraternity and initially
restricted NGS acceptance. It was the high accuracy in reading DNA capability of NGS that
boosted confidence and prompted its widespread use, thus breaking the hegemony of
Sanger’s 30 years domination.
Chasing the message coded by the genotypes, which is directly related to individual
phenotypes, is a humongous task. Improvement of modern NGS instruments, which are
capable of producing millions of DNA sequence reads in a single run, is rapidly changing
our view of genetics, providing the ability to answer questions with unimaginable speed
and huge binary outputs. These modern day instruments provide an inexpensive way to
analyse various applications ranging from chromatin immunoprecipitation, mutation
mapping, polymorphism discovery to noncoding RNA discovery.
In more recent times, NGS has moved forward from the conventional laboratory practices
to applied research, and has been proven as essential tool for ongoing pharmaceutical
research. It is capable enough to address general and specialized genomic question of
mutagenesis, changes in gene expression behaviour, gene regulatory mechanisms, for
population-based studies of communicable and non-communicable diseases.
NGS is capable of
addressing general and
specialized genomic
question of
mutagenesis, changes
in gene expression
behaviour, gene
regulatory
mechanisms, for
population-based
studies of
communicable and
non-communicable
diseases.
“
“
DIVERSE CLINICAL APPLICATIONS: Higher accuracy, greater depth and wider coverage of NGS enable clinicians to
prepare a molecular landscape of disease initiation, progression and establishment along with the therapeutic impact of
drug candidate at gene expression level. It provides wide range of assays to serve specific needs of projects. Major category of
assays along with their specific purpose are:
Wholegenomesequencing generates a complete genomic portfolio of individual
Targetsequencing and Exome-Seq provide information for a subset of genome region
RNA-Seq provides quantification value of gene expression and changes in the gene expression under a disease condition
or a drug exposure
ChIP-Seq provides gene regulatory mechanism involved in the change of gene expression due to occurrence of disease
or drug treatment
Traditionally, NGS experiments generate terabytes of data in the form of text files. To decipher meaningful information from
this raw data is like seeking a needle in a haystack. Obviously, this requires high–end computing (in terms of storage, retrieval
and management) and analytic algorithms. Here, bioinformatics plays a crucial role in raw data processing, quality controls
and analytics.
Bioinformatics has, thus, become an important component in clinical or diagnostic laboratories for generating, analysing,
maintaining, and interpreting data from molecular genetics testing. Given the rapid adoption of NGS-based testing, Incedo
developed informatics work flows, that adhere to the rigor of laboratory standards, yet are flexible to changes as the
chemistry and software for analysing sequencing data mature.
1. Human Genome Project Archive 1990 – 2003
http://web.ornl.gov/sci/techresources/Human_Genome/project/budget.shtml
2. http://www.genome.gov/10001772
3. http://www.genome.gov/pages/der/sequencing_costs_apr2015.xlsx
4. Lecuit M, Eloit M. The diagnosis of infectious diseases by whole genome next generation sequencing: a new era is opening.
Frontiers in Cellular and Infection Microbiology. 2014;4:25. doi:10.3389/fcimb.2014.00025.
5. He, M. et al. Emergence and global spread of epidemic healthcare-associated Clostridium difficile. Nature Genet. 45, 109–113
(2013).
6. Mellmann, A. et al. Prospective genomic characterization of the German enterohemorrhagicEscherichia coli O104:H4 outbreak
by rapid next generation sequencing technology. PLoS ONE 6, e22751 (2011).
7. Rohde, H. et al. Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N. Engl J. Med. 365, 718–724 (2011).
8. Prosperi, M. et al. Molecular epidemiology of community-associated methicillin-resistantStaphylococcus aureus in the
genomic era: a cross-sectional study. Sci. Rep. 3, 1902(2013).
9. Gardy, J. L. et al. Whole-genome sequencing and social-network analysis of a tuberculosis outbreak. N. Engl. J. Med. 364,
730–739 (2011).
10. Lisa H. et al. Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers.
Modern Pathology , (31 July 2015) | doi:10.1038/modpathol.2015.86
11. Wong E.S.Y. et al. Predictive Factors for BRCA1 and BRCA2 Genetic Testing in an Asian Clinic-Based Population. PLoS ONE
10(7): e0134408 (2015). doi:10.1371/journal.pone.0134408
12. Meldrum C. et al. . Next-Generation Sequencing for Cancer Diagnostics: a Practical Perspective. The Clinical Biochemist
Reviews. 2011;32(4):177-195.
13. Banerji, S. et al., Sequence analysis of mutations and translocations across breast cancer subtypes. Nature, 2012, 486(7403),
405–409.
14. Ding, L. et al., Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature, 2012,
481(7382), 506–510.
15. Agrawal, N. et al., Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1.
Science, 2011, 333(6046), 1154–1157.
REFERENCE
A WAY FORWARD:
http://www.incedoinc.com/lifesciences/researchanddevelopment
Reduction in the cost of NGS is unrivalled and has made it accessible to many. It has raised the
hopes and aspirations to answer mysteries and to conduct very large set studies on various diseases, which remained
a distant dream under Sanger sequencing technology. However, data deluge is a major bottleneck for data analysis. Data
management, analysis and interpretation have always been a challenge for the scientific community and statisticians alike.
At Incedo, we have examined bioinformatics software available for whole Exome data analysis, including data pre-
processing, alignment, post-alignment processing, and variant calling, annotation, and prioritization tools. Also,
performance of alignment tools and variant calling programs has been benchmarked, using simulated and sample datasets,
for a perfect precision. Overall, we strongly believe that deployment of various NGS methods at different molecular level for
large scale patient's population will provide much wider and deeper molecular understanding for systemic progression of a
disease or drug response.
For more information about Incedo’s bioinformatics analytic offerings, visit:
White Paper
Incedo Inc. (formerly a part of $ 4Bn Indiabulls Group) is a technology services and outsourcing organization
headquartered in the Bay Area, USA with workforce across North America, South Africa and India (Gurgaon, Bangalore).
We specialize in Data & Analytics and Product Engineering Services, with deep expertise in Financial Services, Life
Science and Communication Engineering. Our key focus is on Emerging Technologies and Innovation. Our end-to-end
capabilities span across Application Services, Infrastructure and Operations.
INDIA: | 248, Udyog Vihar Phase-IV, Gurgaon - 122 015 | Tel: +91 124 4345900/01/02
USA: | 2350 Mission College Boulevard, Suite 246 Santa Clara, California - 95054 | Tel: +1408 531 6040
GOPAL JOSHI
ATEEQ KHALIQ
NIDHI CHAUHAN
Scientist - Bioinformatics | Pharma and Life Sciences Practice
Email: gopal.joshi@incedoinc.com
https://in.linkedin.com/in/gopsa
Scientist - Bioinformatics | Pharma and Life Sciences Practice
Email: ateeq.khaliq@incedoinc.com
https://in.linkedin.com/in/ateeqkhaliq
Manager – Presales
Email: nidhi4@incedoinc.com
https://www.linkedin.com/in/chauhannidhi

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Next generation sequencing

  • 1. White Paper INTRODUCTION: Human genome sequencing project completion is considered as a major milestone in genomic research. This global effort had established a new vision of advance therapeutics. It was mainly accomplished by conventional ‘Sanger Sequencing’ method, which had taken rigorous efforts of lab workers for 12 years and approximately $3 billion were spent. On similar lines, Prof. Craig Venter‘s lab also sequenced a human genome. However, successful completion of these two competing human genome projects could not satisfy biologists’ hunger for greater sequencing throughput and more economical sequencing. The main limitations of Sanger method were high cost, low speed and labour intensive nature. Due to these limitations, the Sanger sequencing process could not fulfill the rapid increase in demand from pharma industries and genomic research facilities. This vacuum was successfully filled by next generation sequencing methods, as they are cost effective, less time consuming and capable enough to read millions of DNA sequences in parallel. NGS methods are cost effective, less time consuming and capable enough to read millions of DNA sequences in parallel. “ “ Next generation sequencing- advancement in Pharmaceutical Research
  • 2. White Paper According to recent facts from National Human Genome Research Institute, an estimated cost for the human genome sequencing project is ~$4211. This signifies a greater reduction in cost when compared with first human genome sequencing. Credit, for this, goes to advancement in sequencing methodologies i.e. parallel reading of DNA sequences. Considering the reading depth and large coverage capabilities of NGS methods a more ambitious `1000 genome sequencing project’ has been accomplished. The main aim of this project is to find all possible mutations in human genome, which might be responsible for a disease condition. This data repository of 1000 genome project is growing rapidly. In fact, a recent report indicates accomplishment of 2504 human genomes for the same. Clearly, the growth of genomic data in the biological repository is in agreement with the advancement of NGS technology. NGS IN PHARMA RESEARCH: Non-Sanger based sequencing technologies provided a tremendous boost to the scientific community, thereby enabling them to discover novel facts and achievements. This changed their way of thinking, thus providing deeper insights into our genetic makeup. However, the significant contribution of Sanger sequencing had created a comfort zone within the scientific fraternity and initially restricted NGS acceptance. It was the high accuracy in reading DNA capability of NGS that boosted confidence and prompted its widespread use, thus breaking the hegemony of Sanger’s 30 years domination. Chasing the message coded by the genotypes, which is directly related to individual phenotypes, is a humongous task. Improvement of modern NGS instruments, which are capable of producing millions of DNA sequence reads in a single run, is rapidly changing our view of genetics, providing the ability to answer questions with unimaginable speed and huge binary outputs. These modern day instruments provide an inexpensive way to analyse various applications ranging from chromatin immunoprecipitation, mutation mapping, polymorphism discovery to noncoding RNA discovery. In more recent times, NGS has moved forward from the conventional laboratory practices to applied research, and has been proven as essential tool for ongoing pharmaceutical research. It is capable enough to address general and specialized genomic question of mutagenesis, changes in gene expression behaviour, gene regulatory mechanisms, for population-based studies of communicable and non-communicable diseases. NGS is capable of addressing general and specialized genomic question of mutagenesis, changes in gene expression behaviour, gene regulatory mechanisms, for population-based studies of communicable and non-communicable diseases. “ “ DIVERSE CLINICAL APPLICATIONS: Higher accuracy, greater depth and wider coverage of NGS enable clinicians to prepare a molecular landscape of disease initiation, progression and establishment along with the therapeutic impact of drug candidate at gene expression level. It provides wide range of assays to serve specific needs of projects. Major category of assays along with their specific purpose are: Wholegenomesequencing generates a complete genomic portfolio of individual Targetsequencing and Exome-Seq provide information for a subset of genome region RNA-Seq provides quantification value of gene expression and changes in the gene expression under a disease condition or a drug exposure ChIP-Seq provides gene regulatory mechanism involved in the change of gene expression due to occurrence of disease or drug treatment Traditionally, NGS experiments generate terabytes of data in the form of text files. To decipher meaningful information from this raw data is like seeking a needle in a haystack. Obviously, this requires high–end computing (in terms of storage, retrieval and management) and analytic algorithms. Here, bioinformatics plays a crucial role in raw data processing, quality controls and analytics. Bioinformatics has, thus, become an important component in clinical or diagnostic laboratories for generating, analysing, maintaining, and interpreting data from molecular genetics testing. Given the rapid adoption of NGS-based testing, Incedo developed informatics work flows, that adhere to the rigor of laboratory standards, yet are flexible to changes as the chemistry and software for analysing sequencing data mature.
  • 3. 1. Human Genome Project Archive 1990 – 2003 http://web.ornl.gov/sci/techresources/Human_Genome/project/budget.shtml 2. http://www.genome.gov/10001772 3. http://www.genome.gov/pages/der/sequencing_costs_apr2015.xlsx 4. Lecuit M, Eloit M. The diagnosis of infectious diseases by whole genome next generation sequencing: a new era is opening. Frontiers in Cellular and Infection Microbiology. 2014;4:25. doi:10.3389/fcimb.2014.00025. 5. He, M. et al. Emergence and global spread of epidemic healthcare-associated Clostridium difficile. Nature Genet. 45, 109–113 (2013). 6. Mellmann, A. et al. Prospective genomic characterization of the German enterohemorrhagicEscherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS ONE 6, e22751 (2011). 7. Rohde, H. et al. Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N. Engl J. Med. 365, 718–724 (2011). 8. Prosperi, M. et al. Molecular epidemiology of community-associated methicillin-resistantStaphylococcus aureus in the genomic era: a cross-sectional study. Sci. Rep. 3, 1902(2013). 9. Gardy, J. L. et al. Whole-genome sequencing and social-network analysis of a tuberculosis outbreak. N. Engl. J. Med. 364, 730–739 (2011). 10. Lisa H. et al. Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers. Modern Pathology , (31 July 2015) | doi:10.1038/modpathol.2015.86 11. Wong E.S.Y. et al. Predictive Factors for BRCA1 and BRCA2 Genetic Testing in an Asian Clinic-Based Population. PLoS ONE 10(7): e0134408 (2015). doi:10.1371/journal.pone.0134408 12. Meldrum C. et al. . Next-Generation Sequencing for Cancer Diagnostics: a Practical Perspective. The Clinical Biochemist Reviews. 2011;32(4):177-195. 13. Banerji, S. et al., Sequence analysis of mutations and translocations across breast cancer subtypes. Nature, 2012, 486(7403), 405–409. 14. Ding, L. et al., Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature, 2012, 481(7382), 506–510. 15. Agrawal, N. et al., Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science, 2011, 333(6046), 1154–1157. REFERENCE A WAY FORWARD: http://www.incedoinc.com/lifesciences/researchanddevelopment Reduction in the cost of NGS is unrivalled and has made it accessible to many. It has raised the hopes and aspirations to answer mysteries and to conduct very large set studies on various diseases, which remained a distant dream under Sanger sequencing technology. However, data deluge is a major bottleneck for data analysis. Data management, analysis and interpretation have always been a challenge for the scientific community and statisticians alike. At Incedo, we have examined bioinformatics software available for whole Exome data analysis, including data pre- processing, alignment, post-alignment processing, and variant calling, annotation, and prioritization tools. Also, performance of alignment tools and variant calling programs has been benchmarked, using simulated and sample datasets, for a perfect precision. Overall, we strongly believe that deployment of various NGS methods at different molecular level for large scale patient's population will provide much wider and deeper molecular understanding for systemic progression of a disease or drug response. For more information about Incedo’s bioinformatics analytic offerings, visit: White Paper
  • 4. Incedo Inc. (formerly a part of $ 4Bn Indiabulls Group) is a technology services and outsourcing organization headquartered in the Bay Area, USA with workforce across North America, South Africa and India (Gurgaon, Bangalore). We specialize in Data & Analytics and Product Engineering Services, with deep expertise in Financial Services, Life Science and Communication Engineering. Our key focus is on Emerging Technologies and Innovation. Our end-to-end capabilities span across Application Services, Infrastructure and Operations. INDIA: | 248, Udyog Vihar Phase-IV, Gurgaon - 122 015 | Tel: +91 124 4345900/01/02 USA: | 2350 Mission College Boulevard, Suite 246 Santa Clara, California - 95054 | Tel: +1408 531 6040 GOPAL JOSHI ATEEQ KHALIQ NIDHI CHAUHAN Scientist - Bioinformatics | Pharma and Life Sciences Practice Email: gopal.joshi@incedoinc.com https://in.linkedin.com/in/gopsa Scientist - Bioinformatics | Pharma and Life Sciences Practice Email: ateeq.khaliq@incedoinc.com https://in.linkedin.com/in/ateeqkhaliq Manager – Presales Email: nidhi4@incedoinc.com https://www.linkedin.com/in/chauhannidhi