1. Celera HIV Genotyping using the Qiagen Q24 Pyromark:
An Alternative to Sanger Sequencing?
A Feasibility Study
Robert Bruce
Scientist, Product Development
Celera
December 3, 2014
2. Purpose of the Study
The objective of the study was to determine if pyrosequencing was a viable
alternative to traditional Sanger sequencing as a method for drug resistance
genotyping of HIV-1. Pyrosequencing has the potential advantages of
accuracy, flexibility, parallel processing, and can be automated. Furthermore,
the technique dispenses with the need for labeled primers, labeled
nucleotides, and gel-electrophoresis with the additional advantages of higher
throughput and lower cost.
2
3. 3
What is Pyrosequencing?
Pyrosequencing
● A method of DNA sequencing (determining the order of nucleotides in DNA)
based on the “sequencing by synthesis" principle.
● It differs from Sanger sequencing, relying on the detection of pyrophosphate
release (hence the name) on nucleotide incorporation, rather than chain
termination with dideoxynucleotides.
4. 4
Pyrosequencing Reaction Cascade
● ssDNA template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP
sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.
● The addition of one of the four deoxynucleotide triphosphates (dNTPs)(in the case of dATP we add dATPαS which
is not a substrate for a luciferase) initiates the second step. DNA polymerase incorporates the correct,
● complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.
● ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts
as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that
are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a
camera and analyzed in a program.
● Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another
nucleotide. This is illustrated in the following schematic:
5. 5
Why Consider a Pyrosequencing Approach to HIV Resistance
Genotyping?
Quicker method than conventional Sanger sequencing.
Advantages of Pyrosequencing
NO thermocycling
NO post sequencing cleanup
NO lengthy instrument set up
NO pull-up due to high signal
NO need for a dye matrix
NO need for a mobility file
NO uneven peak height
NO anomalies due to kinetics of dye terminator incorporation.
Can be run in as little as 30 minutes after PCR resulting in higher throughput
6. 6
Schematic of HIV-1 Protease Gene Showing Placements of PCR and
Sequencing Primers
7. 7
Details of Assay Design
Two PCR setups for amplification
Only the PCR reverse primer was biotinylated
Primer was placed in conserved regions
One amplification covered the complete protease and rt region (1.3 kb)
The second Amplification covered only the protease region (356 b)
Three sequencing primers were designed to sequence the regions
containing the mutations of interest.
8. 8
HIV Mutations Selected for this Study
Two HIV plasmids (wild type and mutant) encoding the protease and reverse
transcriptase genes were used.
Only protease region was sequenced for this feasibility study.
Mutations covered in the protease gene were:
● – D30N GAT AAT
● – M46I ATG ATA
● – G48V GGG GTG
● – I50V ATT GTT
● – V82F GTC TTC
● – I84V ATA GTA
● – L90M TTG ATG
These mutations appear early in the evolution of inhibitor resistance and are
the major primary resistance mutations, inhibiting the binding of the
inhibitor to the protease,
9. Materials and Methods and Procedure
Starting Material
● Wild type and mutant plasmids.
● Mixtures of plasmids containing a range of variant content.
Method
● PCR of plasmids was done using the PyroMark PCR kit
Procedure
● To detect and quantitate mixed bases.
● Use pre-determined dispensation order to detect known variants at specific
codons.
● Quantitative analysis done using AQ analysis software to demonstrate
detection of mixed bases and linearity of measurement.
● Compare with Sanger sequencing
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12. 12
Comparison of 1.3 kb and 356 b Results at Codon 30
G A T (A A ) T
356 bp
1.3 kb
1.3 kb
13. 13
Pyrosequencing Protocol Summary
● All experiments were done on a Q24 Pyromark Instrument following the
handbook protocol
● In all following experiments the 356bp amplicon was used
● All assays were done in AQ (Allele Quantitation) which automatically analyzes
mutation levels.
● Nucleotide dispensation orders were initially pre-defined as “Sequence To
Analyze” and adjusted manually if required. Mutations were noted by mixed
base calls. In these regions, the appropriate nucleotides were added in
sequence. (ie.)
M=A,C
R= G,A
Y=C,T
K=G,T
S=G,C.
For example, codon 30 (D30N) would be defined as R A T, and the G and A
nucleotides would be added.
14. 14
Results of Codon 30 Assay with 356 bp Amplicon
D30N GAT AAT
Wild Type
Mutant
G A T
(AA) T
15. 15
Results of Codons 46-50 Assay with 356bp Amplicon :
M46I ATG ATA
G48V GGG GTG
I50V ATT GTT
Wild Type
Mutant
16. 16
Results of Codons 46-50 Assay with 356bp Amplicon :
M46I ATG ATA
G48V GGG GTG
I50V ATT GTT
Wild Type
Mutant
17. 17
Results of Codons 46-50 Assay with 356bp Amplicon :
M46I ATG ATA
G48V GGG GTG
I50V ATT GTT
Wild Type
Mutant
18. 18
Result of Codons 82-90 Assay with 356 bp Amplicon:
V82F GTC TTC
I84V ATA GTA
L90M TTG ATG
Wild Type
Mutant
19. 19
Result of Codons 82-90 Assay with 356 bp Amplicon:
V82F GTC TTC
I84V ATA GTA
L90M TTG ATG
Wild Type
Mutant
20. 20
Result of Codons 82-90 Assay with 356 bp Amplicon:
V82F GTC TTC
I84V ATA GTA
L90M TTG ATG
Wild Type
Mutant
24. 24
Linearity of ViroSeq Kit using Sequencing Mixes A, D and F
(These are the mixes used to sequence the protease region)
using Mutation Surveyor.
25. 25
Sequencing Codons 30-50 in One Read (WT, 80 bp)
● Initial experiment looks promising
● Optimization needed to allow sensitive quantification
● 5-A and 5-G homopolymers at dispensations 34 and 44 are resolved
~without shift problems
● signal drop-off from 25,68 (T at disp2) to 16,36 (T at disp57) 36%
Initial experiment looks promising.
Optimization needed to allow sensitive quantification.
5-A and 5-G homopolymers at dispensations 34 and 44 are resolved.
Signal drop-off as read length increases.
G A T A T G GGG ATT
5 A 5 G
26. 26
Cyclic dispensation in AQ mode
Cyclic dispensation in AQ mode is an option to cover unexpected mutations
Quantification gets difficult in late positions (slight increase in noise)
Works best with a pure sample (not a mixture)
27. Comparison of AQ mode (4 nucleotides/cycle) with Sanger Sequencing
27
T G A T A C A G TATT A GAA G AAA TGA
pyrogram
Sequence translation from pyrogram
electropherogram
28. 28
Summary
● Three Pyrosequencing assays to quantify mutations in Codons 30, 46, 48,
50, 82, 84 and 90 of the HIV protease gene were designed and tested
1 PCR amplicon (356 bp)
3 Sequencing primers
● Quantification of mixed bases for all three assays demonstrated
good linearity
low variability
sensitivity probably <5% for most mutations
● Short read lengths due to gradual inhibition of ayprase leading to
background peaks and reduced light signals in the sequencing reaction.
● Difficulty in sequencing large amplicons (low peak height, high background)
(Qiagen has updated the chemistry and algorithms so read lengths have
increased.)
