Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Abstract

BackgroundGreat efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance.Methods/ResultsWe have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples.ConclusionThe developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3–5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

“…The Pyrosequencing methodology is a synthesis process, typically, a "wash-and-scan" technique, where the DNA polymerase halts between read steps and may introduce mutation errors. Furthermore, It has been shown that mutation errors in pyrosequencing reads tend to occur predominantly in homopolymeric regions (17,27). In the present study we did not assess the error rate of the present assay; however, others have estimated the overall error rate of the pyrosequencing technique to be on the average of 0.3% (12,28).…”

“…In the present study we did not assess the error rate of the present assay; however, others have estimated the overall error rate of the pyrosequencing technique to be on the average of 0.3% (12,28). It was also previously shown that the pyrosequencing error rate for detection of 17 known drug resistance mutations, located at homopolymer regions in the PR and RT genes of subtype B viruses, was Ͻ0.71% (17). Nevertheless, it was recently reported that the RT K65R mutation that is located at a homopolymer region of subtype C viruses had a pyrosequencing error rate of detection of up to 1.3% (27).…”

“…Furthermore, it was also demonstrated that these low-abundance mutations can have a significant impact on virological failure, mostly for non-nucleoside analog RT inhibitor (NNRTI) mutations (6,(15)(16)(17)(18). However, despite its high accuracy and effectiveness, the GS FLX platform exceeds the capacity of smalland medium-scale projects, limiting its application for routine testing.…”

“…Recently, Roche introduced a benchtop version of this sequencing platform, the GS Junior (GSJ) System, which is scaled to accommodate fewer samples. Currently, in-house HIV genotypic tests have been developed for this platform (16,17,19); however, the lack of a standardized commercial kit limits the wide-use application of this system in clinical settings.…”

Análisis genético de las mutaciones presentes en las poblaciones virales en pacientes con infección por VIH-1 en Ecuador

“…Luego se procedió a la transcripción reversa, síntesis de ADN complementario, generación de amplicones, purificación de los productos de la reacción de polimerasa en cadena (RPC), cuantificación de los productos purificados de RPC, elaboración de la librería de amplicones, RPC en emulsión, recuperación de las perlas con los productos de RPC obtenidos, cuantificación de las perlas recuperadas y la secuenciación, utilizando las especificaciones detalladas en el manual GS Junior Training (Roche) 8 y las instrucciones del fabricante (Roche) 9,10 . En 27 muestras se utilizó la versión 2.0 que permite la detección de fragmentos del gen pol que codifican para la transcriptasa reversa (TR) y la proteasa (PR) con un límite de detección de 20.000 copias/mL 11 , en el resto de las muestras (74 plasmas) se utilizó la versión 3.0 en la que además de los fragmentos anteriores se detecta el fragmento que codifica la integrasa (IN) y posee un límite de detección de 2.000 copias/mL 12 .…”