About application of NGS technique in Virus disease diagnosis

1. Next Generation Sequencing-
A promising technique for virus
disease diagnosis
Presented by-
Eben Titus E
M-6160
Veterinary Microbiology
Major Credit Seminar

2.  History of sequencing
 Types of NGS System
 1st Gen sequencing
 2nd Gen sequencing
 NGS workflow
 Steps in NGS sequenicng
 NGS Application in Virology
 Quasispecies
 Anti-Viral Drug Resistance
 Novel genotype identification
 Quality control of Live vaccines
 Conclusion and Perspectives
 References
Overview

3. History of Sequencing
Nucleic Acid Sequencing
It is a method to figure out the
exact order of nucleotides present
in the DNA/RNA sample
(Grada et al., 2013)
Nguyen et.al. 2018

4. Types of NGS System

5. Sanger Sequencing
•Developed – 1977
•Chain Termination sequencing/Dideoxy sequencing
•ddNTP (Fluorescently or radioactively labelled) lack 3-OH
group
(Gupta et.al., 2019) Fred Sanger
1st Generation sequencing

6. •High Throughput
Sequencing(Rapidity)
•Massively Parallel Sequencing
•Reduced sample size, low reagent
cost and less time.
Illumina MiSeq Applied Biosystem – SOLID
5500
Roche – 454 pyrosequencing
Commercially available technologies:
1. Illumina/Solexa
2. Roche/454
3. Life/APG – SOLiD system
4. Ion Torrent technology
5. Helicos Biosciences
6. Pacific Biosciences (Gupta et.al.,2019)
2nd (Next) Generation sequencing

7. Cynthia et al., 2019
NGS workflow

8. Step 1-Adapter Binding

9. Ahmed et al., 2019
Step 2- Cluster Amplification(Bridge PCR)

10. Step 2-Cluster Amplification (Emulsion PCR)

11. Step 3 – Sequencing and Imaging (pyrosequening – Roche 454)

12. Step 3-Sequencing and Imaging (Reversible Termination Sequenicng – Illumina)

13. Chang et al., 2015
Step 3-Sequencing and Imaging (Ion Torrent sequening)

14. 1. Quality Control Check(Error
correction)
2. Mapping and Alignemnt (Consensus
sequence identification)
3. Variant detection (SNV, InDel and
CNV)
https://genomehubcam.wordpress.com/ngs-data-2/
Step 4(Data Analysis and Interpretation)

15.  Removal of adaptors
 Demultiplexing
 Removal of Low Quality Sequence
 Screening and Removal of host
sequences
Patil et. al., 2012
STEP 4(DATA ANALYSIS AND INTERPRETATION) QUALITY CONTROL OF QUERY SEQUENCE

16. https://training.galaxyproject.org/training-
material/topics/
Can be used if closely related genomic
sequence is present.
STEP 4(DATA ANALYSIS AND INTERPRETATION) MAPPING AND ALIGNMENT
 Can be used if no knowledge about the
correct sequence/order of reads is known
https://thesequencingcenter.com/knowledge-
base/de-novo-assembly/
Reference Genome Mapping De novo Assembly Mapping

17. Single Nucleotide Variation InDel mutations
STEP 4(DATA ANALYSIS AND INTERPRETATION) VARIANT DETECTION
https://www.garvan.org.au/research/kinghorn-centre-
for-clinical-genomics/learn-about-genomics/for-
gp/genetics-refresher-1/types-of-variants

18.  Full length Viral Genome Sequenicng
 Monitoring anti-viral drug resistance
 Quality control of Live vaccines
 Characterization of Viral quasispecies
 Detection of novel genotype or novel
viruses
 Epidemiology and evolution of virus
infections
 Detection of Tumor virus
NGS APPLICATIONS IN VIRUS DIAGNOSIS
Barzon et.al.2011

19.  Two patients with chronic Hepatitis C
virus was studied
 Treatment – Peg IFN – alpha and Ribavirin
and DAA drugs for 48 weeks for one
patient and other one is treatment naïve
 Sustained Virological Response - <50%
 Decided for Viral genome sequencing by
both sanger method and Illumina deep
sequencing
NGS - Detecting Viral
Quasispecies and Anti-
Viral drug resistance
https://en.wikipedia.org/wiki/Hepatitis_C_virus

20. Region Sanger Sequencing Illumina Deep
Sequencing
Core
Region
(E2)
Susceptible AA
positional changes
Position 70 –
Glutamine to
Arginine
Position 91 –
Methionine
Majority (82%) of the
sequence is same to
sanger method
Minority variants(18%)
Position 91 –
Methionine to Leucine
NS5A
ISDR
No substitution
present
Minority variant (16%)
Position 2220 –
Asparitic acid to Valine
NS3 26 AA positional
change
31 AA positional
change
•Sample results were compared with HCV – J
(Prototype HCV 1B genome)
•Substitution change in amino acid resulting in
conformational changes in active site of drug –
Reduced drug susceptibility and drug
resistance
•Viral Variants – Quasispecies
•Quasispecies – It is a complex distribution of
closely related variants genomes subject to
genetic variation and recombination (Domingo
et.al., 2019)
•NGS Vs Sanger – Identifying the quasispecies
even in lower concentrations.
Ashfaq et.al.2011)

21. NGS - Anti-Viral drug
resistance
Döring et al. Nucleic Acids Research. 2018.
Geno2pheno is the in silico tool
used for detecting Anti-viral drug
resistance for HIV and HCV based NGS
sequences
Changes in the AA sequence
position indicates drug susceptibility
or resistance (NRTI, NNRTI and PI) in
the query sequence

22. Döring et al. Nucleic Acids Research. 2018

23. •Based on Env gp51 – 8 genotypes present worldwide
•Polat et.al., 2016 performed NGS based genome
sequence in cattle suspecting BLV on Peru, Paraguay and
Bolivia(South America)
•Samples were sequenced by Illumina MIseq and aligned
with existing genoypes present.
•Specific mutations were seen in Pol (RT and Integrase),
Env gp51, Tax, Rex and R3 proteins
NGS - Identifying
Novel Genotypes in
viruses
Polat et.al.2017

24. In pol
1. Position 166 – Glutamic acid to Asparitic acid
2. Position 447 – Asparitic acid to Glycine
3. Position 644 – Histidine to Tyrosine
4. Position 826 – Alanine to Threonine
5. Position 792 – Aspargine to Serine (only in
portacheulo samples)
In Env gp51
1. Position 133 – Alanine to Valine

25. In Tax, Rex and R3
1. Position 108 – Phenylalanine to Leucine
2. Position 100 – Proline to Serine (only in
portacheulo samples)
3. Position 113 – Alanine to Glutamic acid
4. Position 26 – Aspargine to histdine
These mutation does not exist with
other genotypes
A new BLV Genotype 9 was
identified in Bolivia (South America)
and reported. (Polat et.al., 2016)

26. Sabin Oral polio Vaccine (OPV3)
Most often losses its attenuating mutations and revert
back to neuro-virulent phenotype
 Cause - Nucleotide mutation in position 472 in 5’
Non-coding Region from attenuated to wildtype
NGS – Detecting
Quality control of
live vaccines

27. MAPREC Test
• Mutation Analysis by PCR and Restricted
Enzyme Cleavage
• Conventional Molecular method quantifying
472U(attenuation) to 472C(Mutant)
• Expensive and Time consuming
NGS Vs MAPREC test
• NIBSC along with 8 laboratory worldwide,
compares NGS and MAPREC in predicting
472C change
• In Pearsons Correlation co-efficient, r = 0.996,
suggesting a closer and positive association
between NGS and MAPREC test.

28. Illumina COVID Seq test
https://www.fda.gov/news-events/press-
announcements/coronavirus-covid-19-update-fda-
authorizes-first-next-generation-sequence-test-
diagnosing-covid-19.
First Antigen based test approved by FDA for detecting
SARS COV2

29. • Earlier, higher cost of sequencing is the barrier for R&D but NGS based
technique has reduced cost to several folds
• Its multi-faceted application in genomics, cancer research, vaccinology,
pharmaco-genomics defines its promptness and soundness as good
alternative in sequencing
• But NGS data analysis and interpretation requires sound computational and
Bio-informatics knowledge – a constrain for others.
• Not withstanding its technical constrains, NGS can be named “ONE TEST FITS
FOR ALL”
Conclusion and Perspectives

30. References
1. Luisa Barzon, Enrico Lavezzo, Valentina Militello, Stefano Toppo and Giorgio Palu.2011. Applications of Next
Generation Sequencing Technologies to Diagnostic Virology. Int. J. Mol. Sci. 2011, 12, 7861-7864.
2. Sunitha M. Kashibhatla, Vaishali P. Waman, Mohan M. Kale M and Urmila Kulkarani – Kale. 2015. Application of
Next generation sequencing data in virology – Opportunities and Challenges.
3. Nidhi Gupta and Vijay K. Verma. 2019. Next-Generation Sequencing and its Application: Empowering in Public
Health Beyond Reality.
4. Meripet Polat, Shin-nosuke Takeshima, Kazuyoshi Hosomichi, Jiyun Kim, Taku Miyasaka, Kazunori Yamada, Mariluz
Arainga. 2016. A new genotype of bovine leukemia virus in South America identified by NGS-based whole
genome sequencing and molecular evolutionary genetic analysis. Retrovirology 2016 13:4
5. Bethany Charlton, Jackson Hockley, Majid Laassri, Thomas Wilton, Laura Crawt, Mark Preston.2020. The use of
Next-Generation Sequencing for the quality control of Live Attenuated Polio Vaccines. JID 2020: 222
6. Mayra Cruz-Rivera, Joseph C Forbi, Lilian H.T. Yamasaki, Carlos A. Vazquez Chacon. 2013. Molecular epidemiology
of viral diseases in the era of Next Generation Sequenicng. Journal of Clinical Virology 57(2013) 378-380
7. L-Na Lu, Clade P Muller, Feng Q He. 2020. Applying Next Generation Sequenicng to unravel the mutational
landscapes in viral quasispecies. Viral Research 283 (2020) 197963
8. Capobianchi M.R., Giombini E., Rozera G. Next Generation Sequenicng technology in clinical virology