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Hospital Universitari Vall d’Hebron
Institut de Recerca - VHIR
Institut d’Investigació Sanitària de l’Instituto de Salud Carlos III (ISCIII)
Bioinformàtica per la
Recerca Biomèdica
http://ueb.vhir.org/2014BRB
Ferran Briansó
ferran.brianso@vhir.org
20/05/2014
BRIEF OVERVIEW TO
AMPLICON VARIANT ANALYSIS
1. DISCLAIMER
2. BASIC CONCEPTS
3. NGS APPROACH
4. SOURCES OF ERROR
5. CLINICAL RELEVANCE
6. BAD NEWS / GOOD NEWS
5
1
2
3
5
6
PRESENTATION OUTLINE
4
DISCLAIMER1
3Extracted from Dr Kassahn's publicly shared slides (2013)
BASIC CONCEPTS2
4
2
* Being able to characterize viral populations rapidly and
accurately is important for understanding pathogenesis,
interplay between viruses and humoral responses, and the
evolution of drug resistance
* Both HIV-1 and HCV exist as viral quasispecies in a host, i.e.
many distinct viral strains are circulating at any given moment in
time
* NGS has the potential to directly sequence many such strains
* Using multiplexing (multiple samples/run), high throughput
can be achieved
5
2 BASIC CONCEPTS
* Which mutations are real?
- Sequencing error
- Assay error / reproducibility
* What frequency of mutations matter clinically?
* DRAMs = Drug resistance associated mutations
* Cloning/Single genome sequencing generates ~10-100
sequences; how representative is this of the entire population?
* NGS approach obtain 1000s of reads/sample from a single run
6
3 NGS APPROACH
SOURCES OF ERROR
7
4
454 SEQUENCING ERROR RATES
8
4
454 SEQUENCING ERROR RATES
8
4
CLINICAL RELEVANCE
9
5
BAD NEWS / GOOD NEWS1
10
6
BAD NEWS / GOOD NEWS1
11
6

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Brief Overview to Amplicon Variant Analysis (UEB-UAT Bioinformatics Course - Session 3.1 - VHIR, Barcelona)

  • 1. Hospital Universitari Vall d’Hebron Institut de Recerca - VHIR Institut d’Investigació Sanitària de l’Instituto de Salud Carlos III (ISCIII) Bioinformàtica per la Recerca Biomèdica http://ueb.vhir.org/2014BRB Ferran Briansó ferran.brianso@vhir.org 20/05/2014 BRIEF OVERVIEW TO AMPLICON VARIANT ANALYSIS
  • 2. 1. DISCLAIMER 2. BASIC CONCEPTS 3. NGS APPROACH 4. SOURCES OF ERROR 5. CLINICAL RELEVANCE 6. BAD NEWS / GOOD NEWS 5 1 2 3 5 6 PRESENTATION OUTLINE 4
  • 3. DISCLAIMER1 3Extracted from Dr Kassahn's publicly shared slides (2013)
  • 4. BASIC CONCEPTS2 4 2 * Being able to characterize viral populations rapidly and accurately is important for understanding pathogenesis, interplay between viruses and humoral responses, and the evolution of drug resistance * Both HIV-1 and HCV exist as viral quasispecies in a host, i.e. many distinct viral strains are circulating at any given moment in time * NGS has the potential to directly sequence many such strains * Using multiplexing (multiple samples/run), high throughput can be achieved
  • 5. 5 2 BASIC CONCEPTS * Which mutations are real? - Sequencing error - Assay error / reproducibility * What frequency of mutations matter clinically? * DRAMs = Drug resistance associated mutations * Cloning/Single genome sequencing generates ~10-100 sequences; how representative is this of the entire population? * NGS approach obtain 1000s of reads/sample from a single run
  • 11. BAD NEWS / GOOD NEWS1 10 6
  • 12. BAD NEWS / GOOD NEWS1 11 6