HIV Drug Resistance Training
Module 5:
Sequencing Procedures:
Commercial and In-House
1
Topics



Overview of Genotyping Procedures
Comparison of Three Different Procedures
Application of Concepts
2
Objectives



Identify individual lab needs for sequencing.
Identify differences among the procedures.
Based on the differences among the procedures,
identify the procedure that best meets the lab’s
needs.
3
overview of genotyping
procedures
What systems are available for HIV DR genotyping?
What do these systems require?
4
HIV DR Genotyping Systems

US FDA-approved HIV-1 genotyping systems:
– ViroSeq™ HIV-1 genotyping system
– TRUGENE® HIV-1 genotyping system

In-house, or “home-brew” genotyping systems
5
Which Procedures do You Currently Use?
ViroSeq
Viroseq only
Trugene


Trugene only

In-house only
ViroSeq and Trugene

Viroseq and In-House

Trugene and In-house
All three
In-house







6
US FDA-approved Systems

Characteristics:
– Approved for HIV-1 subtype B viral DR genotyping *
– Labor-intensive and very expensive

Requirements:
–
–
–
–
–
Specific instruments
Specimen: EDTA plasma
Viral load level: ≥1000-2000 copies/ml of plasma
Extensive training for technicians
Laboratory Biosafety level II working environment for
sample preparation
* They have been demonstrated to work well for non-B subtypes
as well, although with less certainty around performance
characteristics.
7
comparison of three different
procedures
What are the procedures used by ViroSeq™, TruGene®, and inhouse assays?
What factors should be considered in choosing one procedure over
another?
8
Option One: ViroSeq™ HIV-1 Genotyping

Detects HIV-1 subtype B viral resistance
– in plasma samples
– collected in EDTA tubes
– with a viral load ranging from 2,000 to >750,000
copies/ml.

Genotypes the entire HIV-1 protease coding
region (codons 1-99) and codons 1-335 of
reverse transcriptase.
9
Cycle
Sequencing
using an ABI
9700
thermocycler
Analysis
Purification
and
quantification
of RT-PCR
products
Sequencing
Reverse
transcription
(RT) and
Polymerase
chain reaction
(PCR) to
amplify target
gene region
using an ABI
9700
thermocycler
Purification & Quantification
RNA
extraction
from
plasma in
BSL II hood
RT/PCR
Sample Preparation
ViroSeq™ HIV-1: Genotyping Procedure
Automated
sequence
detection
using an ABI
3100 DNA
genetic
analyzer
10
Generate drug
resistance
report
Review
Phylogenetic
analysis to
detect crosscontamination.
Report
Confirmation of
sequencing
analysis by 2nd
technician
Analysis
Sequence
analysis using
ViroSeq HIV-1
genotyping
system
software v2.7.
Confirmation
Analysis
ViroSeq™ HIV-1: Genotyping Procedure
Review drug
resistance
report and
send the report
back to
requester
11
Option Two: TRUGENE® HIV-1 Genotyping



Semi-automated technique
Covers PR (codons 4-99) and RT (codons 38-248)
Viral RNA extracted from plasma
– Plasma viral load ≥1,000 copies/ml


cDNA amplified in a single-tube RT-PCR reaction
Sequenced with a modified Sanger technique
– Dye-primer (not dye-labeled ddNTPs)
– PR and RT sequences non-contiguous
Semi-automated data analysis, alignment and
interpretation
 Specific chemical waste disposal required
(acrylamide)

12
CLIP™
Sequencing
using an ABI
9700
thermocycler
Electrophoresis
RT-PCR
transcription
and
amplification
using an ABI
9700
thermocycler
Sequencing
Sample
preparation:
Viral RNA
extraction using
QIAamp viral
RNA Mini Kit or
other
commercial kits
RT-PCR
Preparation
TRUGENE® HIV-1: Genotyping Procedure
Detection using
Long-Read
Towers
• Cast acrylamide
gels
• Prepare MicroCel™
cassette and
loading gel onto
Long-Reader
Tower
13
Confirmation
of data
analysis by
2nd person
Generate
genotyping
results
Review
Crosscontamination
detection
using Genetic
Fingerprint
Results
Data
verification
Data Analysis
Sequence Evaluation
TRUGENE® HIV-1: Genotyping Procedure
Review drug
resistance
report and
send report
back to
requester
14
Home-brew/In-house Genotyping Assays


Many home-brew genotyping assays
Differences:
– Detection of HIV-1 viral resistance for non-B subtypes
and circulating recombinant forms (CRFs)
– Specimen type: plasma, dried blood spots (DBS), or
dried plasma/serum spots (DPS/DSS)
– Boundaries of the protease and the reverse
transcriptase regions of the pol gene that are
sequenced
– Sensitivity (viral load requirement)
– Other performance characteristics (validation)
– Cost and turn-around time
15
Example: Home-brew/In-house
Genotyping Procedure
RNA or/and
DNA
extraction
(e.g.
NucliSens
extraction kit
or Qiagen RNA
Mini kit)
Target gene
amplified by
RT-PCR and
nested PCR
(e.g. ABI 9700
thermocycler)
Confirmation
of
amplification,
purification
and
quantification
of PCR
products
Cycle
sequencing
(e.g. ABI 9700
thermocycler)
Automated
sequencing
detection
(ABI 3100 or
3730 genetic
DNA analyzer)
16
Example: Home-brew/In-house
Genotyping Procedure
Sequence data
editing
• e.g. ChromasPro,
Sequencher, etc.
• Edits confirmed by a
2nd person, esp.
mixtures
Look for crosscontamination by
phylogenetic
analysis (e.g.
MEGA 4 or
SQUAT software)
Drug resistanceassociated
mutations are
interpreted using
the Stanford HIV
drug resistance
web site
Resistance report
is reviewed by
supervisor and
sent to requester
17
Comparative Analysis : 1 of 4
US FDA
approved
ViroSeqTM
TRUGENE®
CDC Homebrew assay
Yes
Yes
No
FDA approved for
FDA approved for
HIV-1 subtype subtype B, but may subtype B, but may
also work for non-B also work for non-B
Major group M
viruses, A, B,
C, D F and G
and CRF01-AE
and CRF02-AG
Diversification
possibility
Semi-open system
Closed system
Open system
Cost for major
instruments
$200,000
$90,000
$200,000$275,000
18
Comparative Analysis : 2 of 4
ViroSeqTM
TRUGENE®
CDC Homebrew assay
Cross
contamination
control
UNG system
(destroy PCR
amplicons
containing dUTP)
Built in Genetic
Fingerprint for
sequence analysis
varies
Labor intensity
Moderately high
High
Moderately
high
Sequence data
analysis
Experience needed
Semi-automatic
Lots of
experience
needed
Instrument
Maintenance cost
expensive
moderate
expensive
19
Comparative Analysis : 3 of 4
TRUGENE®
CDC Homebrew assay
System specific
Non-system
specific
software
BSL II cabinet
BSL II cabinet and
Specific chemical
disposal requirement
BSL II cabinet
Biohazard waste
generated
Moderate
High including
specific requirement
for handling chemical
waste
Moderate
Reliability of
supplies
Varies
Varies
Varies
ViroSeqTM
Sequence data
System specific
analysis software
Biosafety
requirement
20
Comparative Analysis : 4 of 4
ViroSeqTM
Labor intensity
(sequence
detection)
Overall
complexity
Cost-effective
consideration
for use
TRUGENE®
CDC Homebrew assay
Heavy, individual
Moderate, batch
Moderate, batch run patient sequence
run
run
Complex
Surveillance and
medium to large
population-based
genotyping services
Complex
Very complex
Small to medium
patient
genotyping
services
Surveillance and
medium to large
populationbased
genotyping
services
21
Cost Comparison
ViroSeq
TRUGENE®
In House
(CDC)
Cost/kit
$5688
$4292
N/A
No. tests/kit
48
30
N/A
Cost/test
$120
$150
$80-150
Actual Cost/test
(including 15%
repeats)
$140
$165
$95-160
22
Summary of HIV-1 Genotyping Procedures
Two US FDA-approved genotyping systems
Many home-brew genotyping systems
All are conventional viral population-based
sequencing assays with a minor viral population
detection limit of ≥20%
 Complex assays - need highly skilled staff,
dedicated instrumentation and infrastructure
 Test and instrument maintenance are expensive



23
application of concepts
Which type of procedure is most appropriate for our national lab?
24
Discussion
What factors are most important in choosing a
procedure?
 Which procedure (if any) meets your needs best?
 What issues do you need to resolve in order to
obtain and use the procedure you have selected?

25
Reflection


Which procedure will we use (if any)?
What must we do as “next steps?”
26
Summary



Overview of Genotyping Procedures
Comparison of Three Different Procedures
Application of Concepts
27