Feasibility of Using 2′ Terminators and a DNA Polymerase With Reduced Discrimination Against Unconventional

Nucleotides in the ViroSeq HIV Genotyping Assay

Robert BruceDevelopment

Introduction

The purpose of this study was to determine whether the technology based on Roche mutant DNA polymerase (Fribo) and 2′ ribonucleotide terminators represented an improvement in cost and robustness compared to the current BigDye system . To test feasibility, we used the ViroSeq genotyping assay as a model system.

Sequencing Schematic Showing Chain Termination by either ddNTPs or 2′Terminators

2’ terminator (tetraphosphate)

The 2′ phosphate group of the 2′ terminator prevents further extension of the DNA.

The Four Dye Labelled Tetraphosphate Terminators Used in This Study

R6G Adenosine Tetraphosphate R110 Guanosine Tetraphosphate

TAMRA Uridine Tetraphosphate ROX Cytosine Tetraphosphate

All four 2′ terminators in this study were labeled with standard rhodamine dyes.

Rhodamime Dyes vs dRhodamine Dyes

Equivalent Reads of Ribosequencing (top panel) and BDT v1.1 (bottom panel)

Ribosequence

BDT v 1.1BDTv1.1

r

Both sequences give balanced reads with no evidence of problematic regions

Need for Pyrophosphatase in Sequencing Reactions

Pyrophosphatase is a component of BDT sequencing chemistry. The purpose is to prevent the occurrence of pyrophosphorolysis, which can occur when levels of pyrophosphate that are generated in the course of sequencing. When levels of pyrophosphate are sufficently high the DNA polymerase, idling after the addition of an d/ddNTP, can catalyze the reverse reaction. The polymerase used here, does not idle, so the reverse reaction cannot occur. Therefore it is not necessary to add a pyrophosphatase to the sequencing mix.

RiboSequencing with and without Pyrophosphatase with Big Dye Sequencing as Control

RiboSeq with PPase

RiboSeq w/o PPase

BDT v1.1

Removal of pyrophosphatase appears to improve signal with riboterminators.

RiboSeq Sequences Assembled in ViroSeq Genotyping Program

All seven Riboterminator sequences assembled in ViroSeq. Consensus sequencematches reference sequence. Region shows double coverage with bidirectional sequence

Comparison between RiboSequencing and Big Dye Terminator in ViroSeqComparison Between RiboSeq and BDT ViroSeq

ViroSeq 2.7

ViroSeq v2.6

RiboSeq

Comparison Between RiboSeq and BDT ViroSeq

ViroSeq 2.7

ViroSeq v2.6

BigDye v1.1

Both Ribosequencing and BDT determined the same mutations

Genotyping Results between RiboSeq and BDT

Both Riboseq and BDT detected the same 37 mutations

Suggested Next Steps

• Expand study to incorporate other instruments, operators and samples to ensure conclusions of study

• Collect data for the development of mobility files specific for this chemistry to improve automated interpretation

• Use improved terminators in ViroSeq Assay system with POP-6 and the ABI 3100, to accurately benchmark it’s performance against current BigDye Terminator based ViroSeq kit

• Examine/estimate reagent cost for terminators and enzyme with ribo-terminator technology

• Determine if ribo-terminator provides any performance improvement over present system with other templates

• Synthesize 2′ terminators using dichloro-rhodamine dyes to determine if they result in greater sensitivity, less noise and spectral overlap

Conclusions

• These experiments demonstrated that ribosequencing may be a viable alternative to conventional BigDye sequencing. The data showed that ribosequencing had the same fidelity as dideoxy sequencing, and could be used in the ViroSeq assay.

• The dyes and enzymes provided by Roche, performed as advertised and did not require extensive optimization when used with the ABI 3130xl Genetic Analyzer.

• The experiment where pyrophosphatase was removed, suggests that there may be both economic and technical advantages.

• Since the read lengths of ribosequence and the BDT were equivalent, we cannot claim that ribosequencing will give longer and more robust reads. It will be necessary to use different systems to determine whether ribosequencing has special advantages.

• The concentrations of these dyes were equivalent or lower than those of BDT. Further analysis will be necessary to determine if this will lead to a cost savings. The cost of the single enzyme is unknown, but may prove to be less than the multiple enzymes in the BDT system.