Evaluation of miniaturized Illumina DNA preparation protocols for SARS-CoV-2 whole genome sequencing

Pillay, Sureshnee; San, James Emmanuel; Tshiabuila, Derek; Naidoo, Yeshnee; Pillay, Yashodani; Maharaj, Akhil; Anyaneji, Ugochukwu J.; Wilkinson, Eduan; Tegally, Houriiyah; Lessells, Richard; Baxter, Cheryl; Oliveira, Túlio de; Giandhari, Jennifer

doi:10.1371/journal.pone.0283219

Cited by 5 publications

(1 citation statement)

References 25 publications

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“…Nextera XT Despite its complexity, Nextera XT, an Illumina-based library preparation method, provides efficient end-to-end workflows like COVIDSeq [41]. This feature aids in simplifying processing from sample to data analysis, although it demands advanced equipment and infrastructure.…”

Section: Library Preparationmentioning

confidence: 99%

Bridging genomic gaps: A versatile SARS-CoV-2 benchmark dataset for adaptive laboratory workflows

Zufan,

Judd,

Walsh

et al. 2024

Preprint

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Genomic sequencing’s adoption in public health laboratories (PHLs) for pathogen surveillance is innovative yet challenging, particularly in the realm of bioinformatics. Low- and middle-income countries (LMICs) face increased difficulties due to supply chain volatility, workforce training, and unreliable infrastructure such as electricity and internet services. These challenges also extend to high-income countries (HICs) where bioinformatics is nascent in PHLs and hampered by a lack of specialized skills and computational infrastructure. This underlines the urgency for flexible and resource-aware strategies in genomic sequencing to improve global pathogen surveillance. In response to these challenges, the present research was conducted to identify and analyse key variables influencing the quality and accuracy of amplicon sequence data. An extensive benchmark dataset was developed that encompassed a diverse collection of isolates, viral loads, primer schemes, library preparation methods, sequencing technologies, and basecalling models, totalling 750 sequences. This dataset was analysed with bioinformatic workflows selected for varying levels of technical capacity. The evaluation focused on quality metrics, consensus accuracy, and common genomic epidemiological indicators. The analysis uncovers complex interactions between multiple parameters in laboratory and bioinformatic processes. emphasising resource-constrained PHLs, practical guidelines are proposed. Insights from the benchmark dataset aim to guide the establishment of specific laboratory and bioinformatics protocols for amplicon sequencing in these settings. The findings can also be used to guide the creation of specialised training curricula, further advancing genomic equity. The benchmark dataset itself allows laboratories to customise and evaluate workflows, catering to their distinct requirements and capacities. Such a holistic approach is imperative to build the capacity to monitor pathogens worldwide.Author summaryThis study marks a step toward equity in the field of pathogen genomics, especially for resource-constrained PHLs. It develops and evaluates a comprehensive amplicon sequencing benchmark dataset, offering vital insights for PHLs engaged in genomic surveillance. In particular, the study finds that the choice of basecaller model has a minimal impact on the quality and accuracy of consensus sequences derived from ONT data, which is crucial for labs with limited computational resources. It also highlights the effectiveness of longer amplicons in ensuring consistent coverage and reducing amplicon dropouts at higher viral loads. While Illumina remains a gold standard for data quality, the combination of the Midnight primer scheme with ONT’s Rapid library preparation is shown to be a viable alternative, reducing costs, procedural complexity, and hands-on time. The study synthesises these findings into practical guidelines to aid in the development of amplicon sequencing workflows for SARS-CoV-2 with implications for other pathogens.

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Section: Library Preparationmentioning

confidence: 99%

Bridging genomic gaps: A versatile SARS-CoV-2 benchmark dataset for adaptive laboratory workflows

Zufan,

Judd,

Walsh

et al. 2024

Preprint

View full text Add to dashboard Cite

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Automation and miniaturization of high-throughput qPCR for gene expression profiling

Raveendran,

Saeed,

Kalikiri

et al. 2024

SLAS Technology

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A comparison between low-cost library preparation kits for low coverage sequencing

Stewart,

Gibson,

Parsa

et al. 2024

Preprint

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In the fields of human health and agricultural research, low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples. However, a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature. In this study, we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits-the KAPA HyperPlus kit (Roche), the DNA Prep kit (Illumina), and an IDT kit-using 96 human DNA samples. Metrics evaluated included imputation concordance with high-depth genotypes, coverage, duplication rates, time for library preparation, and additional optimization requirements. Despite slightly elevated duplication rates in IDT kits, we find that all four kits perform well in terms of imputation accuracy, with IDT kits being only marginally less performant than Illumina and Roche kits. Laboratory handling of the kits was similar: thus, the choice of a kit will largely depend on (1) existing or planned infrastructure, such as liquid handling capabilities, (2) whether a specific characteristic is desired, such as the use of full-length adapters, shorter processing times, or (3) use case, for instance, long vs short read sequencing. Our findings offer a comprehensive resource for both commercial and research workflows of low-cost library preparation methods suitable for high-throughput low coverage whole genome sequencing.

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Evaluation of miniaturized Illumina DNA preparation protocols for SARS-CoV-2 whole genome sequencing

Cited by 5 publications

References 25 publications

Bridging genomic gaps: A versatile SARS-CoV-2 benchmark dataset for adaptive laboratory workflows

Bridging genomic gaps: A versatile SARS-CoV-2 benchmark dataset for adaptive laboratory workflows

Automation and miniaturization of high-throughput qPCR for gene expression profiling

A comparison between low-cost library preparation kits for low coverage sequencing

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