Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

Batty, Elizabeth M.; Kochakarn, Theerarat; Wangwiwatsin, Arporn; Joonlasak, Khajohn; Huang, Angkana T.; Panthan, Bhakbhoom; Jiaranai, Poramate; Kümpornsin, Krittikorn; Kotanan, Namfon; Manasatienkij, Wudtichai; Watthanachockchai, Treewat; Rakmanee, Kingkan; Jones, Amber; Fernandez, Stefan; Sensorn, Insee; Sungkanuparph, Somnuek; Pasomsub, Ekawat; Klungthong, Chonticha; Chookajorn, Thanat; Chantratita, Wasun

doi:10.1101/2020.06.16.154286

Cited by 7 publications

(4 citation statements)

References 20 publications

(23 reference statements)

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“…For samples requiring immediate attention, e.g. those from patients potentially involved in an active outbreak, adapters were added with the DNA Prep kit (Illumina) as previously described 24 . Amplicons were sequenced on the MiSeq platform, using a Nano 500 cycle kit with v2 chemistry (Illumina) (https://doi.org/10.17504/protocols.io.bnnbmdan).…”

Section: Rapid-turnaround Adapter Addition and Sequencingmentioning

confidence: 99%

Sequencing the pandemic: rapid and high-throughput processing and analysis of COVID-19 clinical samples for 21st century public health

et al. 2021

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Genomic epidemiology has proven successful for real-time and retrospective monitoring of small and large-scale outbreaks. Here, we report two genomic sequencing and analysis strategies for rapid-turnaround or high-throughput processing of metagenomic samples. The rapid-turnaround method was designed to provide a quick phylogenetic snapshot of samples at the heart of active outbreaks, and has a total turnaround time of <48 hours from raw sample to analyzed data. The high-throughput method was designed for semi-retrospective data analysis, and is both cost effective and highly scalable. Though these methods were developed and utilized for the SARS-CoV-2 pandemic response in Arizona, U.S, and we envision their use for infectious disease epidemiology in the 21st Century.

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Section: Rapid-turnaround Adapter Addition and Sequencingmentioning

confidence: 99%

Sequencing the pandemic: rapid and high-throughput processing and analysis of COVID-19 clinical samples for 21st century public health

et al. 2021

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“…The present study does have several limitations. First, the 4 methods were tested on a limited number of European samples, all affiliated to the lineage B1, according to the Pangolin classification (23); the present evaluation should be confirmed in larger studies including other lineages in order to be more representative of the global ecology of this emerging virus.As SARS-CoV-2 sequencing is an expanding market and other methods has been published(24)(25)(26) or are probably under development, the present study is not exhaustive. However, the approaches selected herein are representative of the methods mostly used during pandemics and are easy to implement in a diagnostic laboratory.…”

mentioning

confidence: 99%

Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation

Charre¹,

Ginevra²,

Sabatier³

et al. 2020

Preprint

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Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialized kits including a hybridization capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP) and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples, with mNGS and RVOP producing the most complete genomes. For mid viral loads, 2/8 and 1/8 genomes were incomplete (<99%) with mNGS and both CleanPlex and RVOP, respectively. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques yielding complete genomes for 7/8 samples. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in two samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34-nt deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a default of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer annealing or probe-hybridizing regions and would require regular updates of the technique according to SARS-CoV-2 evolution.

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“…The ARTIC Illumina method, a tiling multiplex PCR approach, was the first that enabled WGS of SARS-CoV-2 using Illumina sequencers [29] . The technique has subsequently been optimized and analysis, albeit in small sample numbers, concluded that it delivers sufficient quality to perform phylogenetic analysis [30] , [31] , [32] . It had been used as targeted and random RT-PCR screening with subsequent sequencing of the population in order to study the spread of SARS-CoV-2 through the community [24] .…”

Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 whole-genome sequencing using reverse complement PCR: For easy, fast and accurate outbreak and variant analysis.

Coolen

Wolters

Tostmann

et al. 2021

Journal of Clinical Virology

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During the course of the SARS-CoV-2 pandemic reports of mutations with effects on spreading and vaccine effectiveness emerged. Large scale mutation analysis using rapid SARS-CoV-2 Whole Genome Sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring transmission and rising levels of mutant strains. Here we report a novel WGS technique for SARS-CoV-2, the EasySeq™ RC-PCR SARS-CoV-2 WGS kit. By applying a reverse complement polymerase chain reaction (RC-PCR), an Illumina library preparation is obtained in a single PCR, thereby saving time, resources and facilitating high-throughput screening. Using this WGS technique, we evaluated SARS-CoV-2 diversity and possible transmission within a group of 173 patients and healthcare workers (HCW) of the Radboud university medical center during 2020. Due to the emergence of variants of concern, we screened SARS-CoV-2 positive samples in 2021 for identification of mutations and lineages. With use of EasySeq™ RC-PCR SARS-CoV-2 WGS kit we were able to obtain reliable results to confirm outbreak clusters and additionally identify new previously unassociated links in a considerably easier workaround compared to current methods. Furthermore, various SARS-CoV-2 variants of interest were detected among samples and validated against an Oxford Nanopore sequencing amplicon strategy which illustrates this technique is suitable for surveillance and monitoring current circulating variants.

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Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

Cited by 7 publications

References 20 publications

Sequencing the pandemic: rapid and high-throughput processing and analysis of COVID-19 clinical samples for 21st century public health

Sequencing the pandemic: rapid and high-throughput processing and analysis of COVID-19 clinical samples for 21st century public health

Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation

SARS-CoV-2 whole-genome sequencing using reverse complement PCR: For easy, fast and accurate outbreak and variant analysis.

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