Next Generation Sequencing:

platforms for drug resistance

testing

Martin Dumer
29.04.2016
 454 Sequencing / Roche
GS Junior System
GS FLX+ System
Illumina (Solexa)
HiSeq Systems Next Generation Sequencing,
Genome analyzer IIx Amplified Single Molecule
MiSeq, MiniSeq, NextSeq Sequencing
Life Technologies
SOLiD 5500 System
SOLiD 5500xl System
Ion Torrent PGM
Proton

Helicos
Helicos Genetic Analysis System
Pacific Biosciences/Roche
PacBio RS II, Sequel Third Generation Sequencing,
Oxford Nanopore Technologies Single Molecule Sequencing
GridION System
MinION
Roche
Genia
Requirements for a routine
diagnostic-capable NGS system

Gain in time or information

easy sample preparation / workflow

acceptable tats

data volume adjusted to diagnostic need

cost effectiveness

ready-to-go bioinformatics

meaningful interpretation (clinical relevance)

Ultra-deep-sequencing

Standard Sanger-sequencing

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Ultra-deep-sequencing
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The players
Ion Torrent PGM
(Personal Genome Machine)

The chip is the machine

Ion Torrent workflow
Microwell on a semiconductor chip
Most important ....

iPod docking station

Specifications
Vela Next Generation Sequencing
NGS automation for the IVD routine laboratory

Key Features
CE-IVD
Ready to use in 2 weeks
from installation
Samples
IT Connectivity
Sample Tracking
Sentosa Sentosa Sentosa Sentosa SQ
Sentosa Link SX101 ST401 SQ301 Reporter Sentosa Link

Control System
Automation
Multi-purpose:
Ready to use Reagents
Sample ID
Actionable Design
4 Validated Template Prep IVD Sequencing, Data QC Result Upload
Download Sample Lysis, Extraction, Open-channel Automated
Materials; Library Prep; Reporting
2 Extraction Kits Open-channel
Data Analysis
Data Reporting
Sentosa SQ HCV/HIV Genotyping Assay
Total Hands on Time: 2,5 hours
Steps Instruments/Software Hands-on Time Instruments Time

1) Extraction Plasma / Serum

extraction
SX101 30 minutes 2 hours 10 minutes
2) Library RT-PCR preparation
preparation

Day 1
Thermal
RT-PCR 5 minutes 2 hours 40 minutes
Cycler

Normalization,
Shearing and SX101 10 minutes 3 hours 10 minutes
ligation
3) Template Emulsion PCR 15 minutes 5 hours 30 minutes
preparation
ST401i
ST401e
ISPs enrichment 15 minutes 35 minutes

Day 2
4) Sequencing
Machine
1 hour
initialization and SQ301 5 hours
sequencing

5) Data analysis
Signal processing SQ

Day 3
NA 4 hours
and variant calling Reporter
Design Concept
Target genes
NS3 NS5A NS5B
HCV Genome

Protease
HIV Genome and RT Integrase
P66 (NNRTI)
Codon 1-335
p51 (NRTI)
Codon 1-250

reverse transcriptase Codon 1-289

Codon 1-99
Integrase
Design Concept
Assay specifications
HIV HCV
Types Plasma & Serum
Volume 0.73 mL 0.53 mL
Pre-treatment N/A
Format 16 (15 samples + 1 system control)
Genotyping coverage Subtypes A - K (+some CRFs) GTs 1a, 1b, 2-6
Analytical sensitivity GTs 1-4: 1,000 IU/mL
2,000 cop/mL
(limit of detection) GTs 5-6: 2,000 IU/mL
Analytical sensitivity 25% frequency at 2,000 cop/mL
not validated yet
(variants calling) 5% frequency at 10,000 cop/mL

Median coverage per 200x for genotyping (NS5B)

1,000x for PR/RT, IN
sample 500x for variant calling

2 Amplicons 3 Amplicons (GTs 1a and 1b)

1: PR: aa 1-99 NS3: aa 1-213 (944 bp)
Amplicons
RT: aa 1-335 NS5A: aa 12-200 (604 bp)
2: IN: aa 1-289 NS5B: aa 337-585 (685 bp)
Vela Diagnostics NGS Workflow
Reporting
HCV Variants Calling
Detection of Genotypes and Drug Resistance Mutation
Vela Assay Drug Vela Assay
Target
Drug Manufacturer Genotype Drug Resistance Mutations Resistance Mutation Sequencing
Gene
Coverage Coverage

boceprevir (FDA
Merck 1b V36M, F43S, T54A, R155K, A156S, A156T, V170A All
approved)
V36A, V36G, V36I, V36L, V36M, T54A, T54S, I132V, R155G,
telaprevir (FDA
Vertex 1a , 1b R155K, R155M, R155T, A156F, A156N, A156S, A156T, A156V, All
approved) D168N, V36M + R155K

Boehringer V36M, T54A, T54S, Q80K, Q80L, Q80N, Q80R, R155K, R155Q,
faldaprevir 1a, 1b All
Ingelheim A156T, A156V, D168A, D168G, D168V

Q41R, F43I, F43S, F43V, Q80H, Q80K, Q80R, R109K, R155K, Genotype 1:
NS3 S122A, S122G, S122R, A156G, A156T, A156V, D168A, D168E, codon 1 to 213
simeprevir Janssen R & D 1b D168H, D168I, D168N, D168T, D168V, D168Y, F43S + Q80R, F43S All
+ D168E, Q80K + R155K, Q80R + R155K, Q80R + D168E, Q80H +
D168E, Q80R + D168A

vaniprevir Merck 1b Q41R, F43S, R155K, A156T, D168Y All

R155K, R155T, R155Q, D168A, D168E, D168G, D168H, D168T,
danoprevir Roche 1a, 1b D168V, D168Y
All

R155K, R155T, R155Q, D168A, D168E, D168G, D168H, D168T,

Paritaprevir Abbott 1a, 1b D168V, D168Y
All

Bristol-Myers Genotype 1:
Y93H, L31V, Y93H, L31V + Y93H, M28T, Q30E, Q30H, Q30R,
NS5A daclatasvir
Squibb
1a, 1b L31M, L31V, P32L, Y93C, Y93H, Y93N, M28T + Q30H
All codon 12 to 200

Sofosbuvir (FDA
Gilead 1a, 1b S282T In development
approved)

Boehringer Genotype 1-6:

NS5B Deleobuvir 1a, 1b P495L, P495S. P495A, P495T, P495Q, P496S, V499A All codon 337 to
Ingelheim
585
vx222 Selleck 1a, 1b L419S, R422K, M423T, M423V All

setrobuvir Anadys 1a, 1b M414T, M414L, G554D, D559G All

HCV Variants Calling
Detection of Genotypes and Drug Resistance Mutation
Vela Assay Drug Vela Assay
Target
Drug Manufacturer Genotype Drug Resistance Mutations Resistance Mutation Sequencing
Gene
Coverage Coverage

boceprevir (FDA
Merck 1b V36M, F43S, T54A, R155K, A156S, A156T, V170A All
approved)
V36A, V36G, V36I, V36L, V36M, T54A, T54S, I132V, R155G,
telaprevir (FDA
Vertex 1a , 1b R155K, R155M, R155T, A156F, A156N, A156S, A156T, A156V, All
approved) D168N, V36M + R155K

Boehringer V36M, T54A, T54S, Q80K, Q80L, Q80N, Q80R, R155K, R155Q,
faldaprevir 1a, 1b All
Ingelheim A156T, A156V, D168A, D168G, D168V

Q41R, F43I, F43S, F43V, Q80H, Q80K, Q80R, R109K, R155K, Genotype 1:
NS3 S122A, S122G, S122R, A156G, A156T, A156V, D168A, D168E, codon 1 to 213
simeprevir Janssen R & D 1b D168H, D168I, D168N, D168T, D168V, D168Y, F43S + Q80R, F43S All
+ D168E, Q80K + R155K, Q80R + R155K, Q80R + D168E, Q80H +
D168E, Q80R + D168A

vaniprevir Merck 1b Q41R, F43S, R155K, A156T, D168Y All

R155K, R155T, R155Q, D168A, D168E, D168G, D168H, D168T,
danoprevir Roche 1a, 1b D168V, D168Y
All

R155K, R155T, R155Q, D168A, D168E, D168G, D168H, D168T,

ABT-450 Abbott 1a, 1b D168V, D168Y
All

Bristol-Myers Genotype 1:
Y93H, L31V, Y93H, L31V + Y93H, M28T, Q30E, Q30H, Q30R,
NS5A daclatasvir
Squibb
1a, 1b L31M, L31V, P32L, Y93C, Y93H, Y93N, M28T + Q30H
All codon 12 to 200

Sofosbuvir (FDA
Gilead 1a, 1b S282T In development
approved)

Boehringer Genotype 1-6:

NS5B Deleobuvir 1a, 1b P495L, P495S. P495A, P495T, P495Q, P496S, V499A All codon 337 to
Ingelheim
585
vx222 Selleck 1a, 1b L419S, R422K, M423T, M423V All

setrobuvir Anadys 1a, 1b M414T, M414L, G554D, D559G All

Reference: http://chipts.ucla.edu/2012/09/11/clinically-relevant-hcv-drug-resistance-mutations-figure-and-tables/
Vela Diagnostics NGS Workflow
Report

Sentosa Reporter

in progress

detected frequencies interpretation of frequencies

(so far interpretation of consensus fasta)
MiSeq sequencing instrument

Illuminas benchtop sequencer

no homopolymer problems
current specification:
~ up to 15 Gb output
2x20mio reads of 2x300 nts length
37h run time (2x250 nts)
MiSeq Personal Sequencing System
Cluster generation and Sequencing-by-Synthesis
Library preparation using
NexteraTM XT tagmentation
Easy library preparation
Fast - less than 20 minutes hands-on time
Only 1ng DNA per sample needed
Up to 4 x 96 indices
Normalization step included
Third generation sequencing
MinION Nanopore technology

Source: Oxford NanoporeTM Technologies

MinION Nanopore technology
MinION Nanopore technology
HIV-1 pol amplicon, 1.35kb
Oxford Nanopore MinION specifications

50 MB in 6 hours (up to 500 MB)

cheap: 900,-
read length: several 1.000 bases
Roche Genia
nanopore sequencing
Pacific Biosciences, PacBio RS II
SMRT (Single Molecule Real Time)
 Pacific Biosciences
SMRT: Single molecule real time sequencing

SMRT-cell

zero-mode waveguide
Introducing Roches Next Sequencer
(Pacific Biosciences Sequel)
First Wave Medical Content
Roche SMRT platform will enable comprehensive clinical
research
Highly accurate, sensitive and
HIV comprehensive sequencing technologies
Resistance may enable better therapeutic
management and selection

HCV Higher resolution typing for

Genotyping improved accuracy in treatment
Resistance decisions
Simplifies transplant genetic
testing by reading
HLA completely through the
Typing complex immunologic
region

Microbial New sequencing technologies allow the clinical

Population application of metagenomics, so pathogens can
identification be identified more rapidly, with a potential of
and improving patient outcomes
Resistance
Drug resistance testing
using Illuminas MiSeq
Experimental setup
HIV genome

PRRT IN ENV

whole genome
Fragmentation
 putting things together

mapping

PRRT IN ENV

Reference sequences
Coverage

~10K to 20K
~10K to 20K

PR/RT ENV
~7500 full V3 loops
Reproducibility
Validation isolation

NGS Sanger

rt-pcr/
pooling
nested pcr

fragmentation &
indexing

sequencing sequencing

analysis analysis

comparison
 Number of mutations not detected by the respective
sequencing approach (Sanger vs NGS and vice versa)
at 2% and 10% NGS minority cutoff

16

14

12

10

2 0 0
0
SANGER PR NGS PR SANGER RT NGS RT

CutOff 10% CutOff 2%

N=111 samples from routine HIV-drug resistance including 30 samples w/w resistance mutations
(PR and/or RT) detected using standard Sanger sequencing
The deeptypeHIV report generator
The deeptypeHIV report generator
Patient
identification
and therapy
data

Scored
mutations
per drug

Detailed
information
mut and wt
frequency

Data modules
The deeptypeHIV report generator
 Upload ...sorted-paired_aaFreqs.csv

Pos. 1
of HIV-1
proteas
e
Muts >10% Muts >2%<10%
Patient
identification
and therapy
data

Scored
mutations
per drug

Detailed
Information on information
coverage mut and wt
frequency

Data modules
 The deeptypeHIV report generator

Summary
data on 2%
and 10%
cutoff
QA
Subtype
 The deeptypeHIV report generator

fastas at 2%
and 10%
cutoff
 The deeptypeHIV report generator

HIV-GRADE
interpretation
at 2% and
10% cutoff
The deeptypeHIV report generator,
integrated HIV-GRADE interpretation
 The deeptypeHIV report generator

Use tick boxes

for
visualization
of resistance
situation:
Susceptible
Limited
susceptibility
Intermediate
Resistant
 The deeptypeHIV report generator

Preconfigured
report
components
available
The deeptypeHIV report
The deeptypeHIV report
Acknowledgments

Bettina Spielberger
Kirsten Becker
Anna Memmer
Nina Engel
Alexander Thielen
Bernhard Thiele