A Novel System Control for Quality Control of Diagnostic Tests Based on Next-Generation Sequencing

Chan, Maurice; Smirnov, A.L.; Mulawadi, Fabianus Hendriyan; Lim, Priscilla; Lim, Wen-Huey; Leong, Siew Meng; Low, Hwee Meng; Yee, Mei Qi; Yeo, Yong Qiang; Zhou, Xin; Lee, Charlie; Huang, Wen; Welebob, Louis; Wu, Mengchu

doi:10.1373/jalm.2016.020131

Cited by 5 publications

(3 citation statements)

References 11 publications

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“…By screening reaction conditions based on concentration of cleaned-up PCR products, we determined that lower primer concentrations and longer annealing/extension time were optimal. Given the low-cost of the assay this step could also be performed alongside standard diagnostic qRT-PCR as a quality control measure to help reveal potential false positives 24 .…”

Section: Introductionmentioning

confidence: 99%

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

et al. 2017

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Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas.

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Section: Introductionmentioning

confidence: 99%

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

et al. 2017

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“…PCR inhibition may affect NGS performance [ 68 , 69 ]. Several concepts to detect PCR inhibition are developed within the field of qPCR that also are potentially useful in NGS.…”

Section: Resultsmentioning

confidence: 99%

Considerations and quality controls when analyzing cell-free tumor DNA

Johansson

Andersson

Filges

et al. 2019

Biomolecular Detection and Quantification

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Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.

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“…The Sentosa SQ HIV-1 Genotyping Assay provides a system control for each run, processed from extraction as the 16th sample. 10 The system control acts as both a positive and negative control. Criteria for system control acceptance include positive control assembly quality (completeness !95.0%, error rate 1.00%, median coverage !200 reads), no template control (HIV reads 50 or 0.0001 Â the number of control amplicon reads), and detection of three low-level, noneHIV-1 variants (range 1.0% to 8.0%).…”

Section: Next-generation Sequencingmentioning

confidence: 99%

Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing

Weber

Volkova

Sahoo

et al. 2019

The Journal of Molecular Diagnostics

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Genotypic antiretroviral drug resistance testing is a critical component of the global efforts to control the HIV-1 epidemic. This study investigates the semiautomated, next-generation sequencing (NGS)based Vela Diagnostics Sentosa SQ HIV-1 Genotyping Assay in a prospective cohort of HIV-1einfected patients. Two-hundred sixty-nine samples were successfully sequenced by both NGS and Sanger sequencing. Among the 261 protease/reverse transcriptase (PR/RT) sequences, a mean of 0.37 drug resistance mutations were identified by both Sanger and NGS, 0.08 by NGS alone, and 0.03 by Sanger alone. Among the 50 integrase sequences, a mean of 0.3 drug resistance mutations were detected by both Sanger and NGS, and 0.08 by NGS alone. NGS estimated higher levels of drug resistance to one or more antiretroviral drugs for 6.5% of PR/RT sequences and 4.0% of integrase sequences, whereas Sanger estimated higher levels of drug resistance for 3.8% of PR/RT sequences. Although the samples successfully sequenced by the Sentosa SQ HIV Genotyping Assay demonstrated similar predicted resistance compared with Sanger, 44% of Sentosa runs failed quality control requiring 17 additional runs. This semi-automated NGS-based assay may aid in HIV-1 genotypic drug resistance testing, though numerous quality control issues were observed when this platform was used in a clinical laboratory setting. With additional refinement, the Sentosa SQ HIV-1 Genotyping Assay may contribute to the global efforts to control HIV-1.

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A Novel System Control for Quality Control of Diagnostic Tests Based on Next-Generation Sequencing

Abstract: Background:We describe a novel system control (SC) implemented in an automated AmpliSeq™-based next-generation sequencing (NGS)

Cited by 5 publications

References 11 publications

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

Considerations and quality controls when analyzing cell-free tumor DNA

Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing

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