PrimerPooler: automated primer pooling to prepare library for targeted sequencing

Brown, Silas S.; Chen, Yun-Wen; Wang, Ming; Clipson, Alexandra; Ochoa, Eguzkine; Du, Ming‐Qing

doi:10.1093/biomethods/bpz015

Cited by 2 publications

(1 citation statement)

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“…While the use of multiplex PCR (mPCR) to amplify multiple DNA fragments in the same reaction provides a simpler solution to reducing the number of reactions 16 , it often leads to a decreased sensitivity, due to complex primer-primer interactions and competition for usage of reagents 17 . Therefore, the establishment of optimal primer pools for multiplex PCR can be challenging and may require many sets of primers as seen with the ARTIC protocol for COVID-19 WGS 18, 19 . The development of amplicon-based WGS should ideally be suitable for multiple NGS platforms, especially in developing countries where the burden of RSV is greatest.…”

Section: Introductionmentioning

confidence: 99%

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

Dong

Deng

Aziz

et al. 2022

Preprint

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Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness. Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse‐transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses. The method requires only two reactions for each sample, which significantly reduces the cost and time compared to other commonly used RSV WGS methods. In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. This new method was tested on 200 clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32. A success rate of 88% with a full coverage for the genome of 99 RSV-A and 77 RSV-B was achieved. This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods.

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Section: Introductionmentioning

confidence: 99%

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

Dong

Deng

Aziz

et al. 2022

Preprint

View full text Add to dashboard Cite

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A Rapid and Scalable Multiplex PCR-Based Next-Generation Amplicon Sequencing Method for Familial Hypercholesterolemia Genetic Screening

Imran,

Arvinden,

Mehanathan

et al. 2024

The Journal of Applied Laboratory Medicine

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Background Familial hypercholesterolemia (FH) is a frequently underdiagnosed genetic disorder characterized by elevated low-density lipoprotein (LDL) levels. Genetic testing of LDLR, APOB, and PCSK9 genes can identify variants in up to 80% of clinically diagnosed patients. However, limitations in time, scalability, and cost have hindered effective next-generation sequencing of these genes. Additionally, pharmacogenomic variants are associated with statin-induced adverse effects in FH patients. To address these challenges, we developed a multiplex primer-based amplicon sequencing approach for FH genetic testing Methods Multiplex primers were designed for the exons of the LDLR, APOB, and PCSK9 genes, as well as for pharmacogenomic variants rs4149056 (SLCO1B1:c.521T > A), rs2306283 (SLCO1B1:c.388A > G), and rs2231142 (ABCG2:c.421C > A). Analytical validation using samples with known pathogenic variants and clinical validation with 12 FH-suspected probands were conducted. Library preparation was based on a bead-based tagmentation method, and sequencing was conducted on the NovaSeq 6000 platform. Results Our approach ensured no amplicon dropouts, with over 100× coverage on each amplicon. Known variants in 2 samples were successfully detected. Further, we identified one heterozygous LDLR (p.Glu228Ter) variant and 2 homozygous cases of LDLR (p.Lys294Ter) and LDLR (p.Ser177Leu) variants in patients. Pharmacogenomic analysis revealed that overall 3 patients may require reduced statin doses. Our approach offered reduced library preparation time (approximately 3 h), greater scalability, and lower costs (under $50) for FH genetic testing. Conclusions Our method effectively sequences LDLR, APOB, and PCSK9 genes including pharmacogenomic variants that will guide appropriate screening and statin dosing, thus increasing both efficiency and affordability.

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PrimerPooler: automated primer pooling to prepare library for targeted sequencing

Cited by 2 publications

References 0 publications

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

A Rapid and Scalable Multiplex PCR-Based Next-Generation Amplicon Sequencing Method for Familial Hypercholesterolemia Genetic Screening

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