Pyrosequencing slide presentation rev3.

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

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.

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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.

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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:

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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

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Schematic of HIV-1 Protease Gene Showing Placements of PCR and Sequencing Primers

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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.

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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,

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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Pyrosequencing Assay Workflow

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QIAxcel Analysis of Amplicons Prior to Sequencing

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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

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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.

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Results of Codon 30 Assay with 356 bp AmpliconD30N GAT AAT

Wild Type

Mutant

G A T

(AA) T

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Results of Codons 46-50 Assay with 356bp Amplicon : M46I ATG ATAG48V GGG GTGI50V ATT GTT

Wild Type

Mutant

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Results of Codons 46-50 Assay with 356bp Amplicon : M46I ATG ATAG48V GGG GTGI50V ATT GTT

Wild Type

Mutant

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Results of Codons 46-50 Assay with 356bp Amplicon : M46I ATG ATAG48V GGG GTGI50V ATT GTT

Wild Type

Mutant

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Result of Codons 82-90 Assay with 356 bp Amplicon: V82F GTC TTCI84V ATA GTAL90M TTG ATG

Wild Type

Mutant

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Result of Codons 82-90 Assay with 356 bp Amplicon: V82F GTC TTCI84V ATA GTAL90M TTG ATG

Wild Type

Mutant

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Result of Codons 82-90 Assay with 356 bp Amplicon: V82F GTC TTCI84V ATA GTAL90M TTG ATG

Wild Type

Mutant

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Demonstration of Linearity in Mixed Base Detection: Codon 30 D30N GAT AAT

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Demonstration of Linearity in Mixed Base Detection: Codons 46-50: M46I ATG ATA G48V GGG GTGI50V ATT GTT

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Demonstration of Linearity in Mixed Base Detection: Codons 82-90V82F GTC TTCI84V ATA GTAL90M TTG ATG

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Linearity of ViroSeq Kit using Sequencing Mixes A, D and F (These are the mixes used to sequence the protease region)

using Mutation Surveyor.

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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

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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)

Comparison of AQ mode (4 nucleotides/cycle) with Sanger Sequencing

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T G A T A C A G TATT A GAA G AAA TGA

pyrogram

Sequence translation from pyrogram

electropherogram

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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.)