Hospital Universitari Vall d’HebronInstitut 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 Briansóferran.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

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PRESENTATION OUTLINE

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DISCLAIMER1

3Extracted from Dr Kassahn's publicly shared slides (2013)

BASIC CONCEPTS2

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* 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

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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

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3 NGS APPROACH

SOURCES OF ERROR

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454 SEQUENCING ERROR RATES

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454 SEQUENCING ERROR RATES

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CLINICAL RELEVANCE

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BAD NEWS / GOOD NEWS1

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BAD NEWS / GOOD NEWS1

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