An optimized, amplicon-based approach for sequencing of SARS-CoV-2 from patient samples using COVIDSeq assay on Illumina MiSeq sequencing platforms

Bhoyar, Rahul C.; Senthivel, Vigneshwar; Jolly, Bani; Imran, Mohamed; Jain, Abhinav; Divakar, Mohit Kumar; Sivasubbu, Sridhar

doi:10.1016/j.xpro.2021.100755

Cited by 17 publications

(19 citation statements)

References 4 publications

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“…The Illumina platform is known for its capacity to handle a large number of samples simultaneously by multiplexing samples and for its high yield of sequences [ 17 ]. The MiSeq sequencing technique is usually used to sequence 96 samples in a single flow cell, depending on the different libraries used [ 7 , 18 ]. In the present study, we optimized the protocol to sequence 384 samples in a single flow cell.…”

Section: Discussionmentioning

confidence: 99%

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Simultaneous SARS-CoV-2 Genome Sequencing of 384 Samples on an Illumina MiSeq Instrument through Protocol Optimization

Mze

Beye

Kacel

et al. 2022

Genes

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In the present study, we propose a high-throughput sequencing protocol using aNextera XT Library DNA kit on an Illumina MiSeq instrument. We made major modifications to this library preparation in order to multiplex 384 samples in a single Illumina flow cell. To validate our protocol, we compared the sequences obtained with the modified Illumina protocol to those obtained with the GridION Nanopore protocol. For the modified Illumina protocol, our results showed that 94.9% (357/376) of the sequences were interpretable, with a viral genome coverage between 50.5% and 99.9% and an average depth of 421×. For the GridION Nanopore protocol, 94.6% (356/376) of the sequences were interpretable, with a viral genome coverage between 7.0% and 98.6% and an average depth of 2123×. The modified Illumina protocol allows for gaining EUR 4744 and returning results of 384 samples in 53.5 h versus four times 55.5 h with the standard Illumina protocol. Our modified MiSeq protocol yields similar genome sequence data as the GridION Nanopore protocol and has the advantage of being able to handle four times more samples simultaneously and hence is much less expensive.

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Section: Discussionmentioning

confidence: 99%

“…Recently, a new protocol for the next-generation sequencing of SARS-CoV-2 RNA using the Illumina MiSeq instrument was described [ 7 ]. Minor modifications were introduced to the Illumina COVIDSeq protocol to sequence 94 SARS-CoV-2-positive samples on a MiSeq instrument.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous SARS-CoV-2 Genome Sequencing of 384 Samples on an Illumina MiSeq Instrument through Protocol Optimization

Mze

Beye

Kacel

et al. 2022

Genes

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“…The authors believed this was probably associated with low genome coverage during sequencing by this sequencer (Román-Reyna et al 2021 ). It may be necessary for the interpretetion the other studies results where sequencing was carried out on different platforms and the data obtained were evaluated in aggregate without taking into account the type of sequencer (e.g., virological studies on COVID-19) (Lu et al 2020 ; Bhoyar et al 2021 ). The above data disclose that the iSeq 100 system may be used to evaluate the bacterial profile of the samples to create an overall picture.…”

Section: Discussionmentioning

confidence: 99%

Comparison of iSeq and MiSeq as the two platforms for 16S rRNA sequencing in the study of the gut of rat microbiome

Salamon

Zapała

Krawczyk

et al. 2022

Appl Microbiol Biotechnol

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Amplicon-based next-generation sequencing (NGS) of the 16S ribosomal RNA (16S) regions is a culture-free method used to identify and analyze Procaryota occurring within a given sample. The prokaryotic 16S rRNA gene contains conserved regions and nine variable regions (V1-V9) frequently used for phylogenetic classification of genus or species in diverse microbial populations. This work compares the accuracy and efficacy of two platforms, iSeq and MiSeq from Illumina, used in sequencing 16S rRNA. The most important similarities and differences of 16S microbiome sequencing in 20 fecal rat samples were described. Genetic libraries were prepared according to 16S Metagenomic Sequencing Library Preparation (Illumina) for the V3 and V4 regions of the 16S. The species richness obtained using iSeq technology was lower compared to MiSeq. At the second taxonomy level (L2), the abundance of taxa was comparable for both platforms. At the L7, the taxa abundance was significantly different, and the number of taxa was higher for the MiSeq. The alpha diversity was lower for iSeq than for MiSeq, starting from the order to the species level. The beta diversity estimation revealed statistically significant differences in microbiota diversity starting from the class level to the species level in samples sequenced on two investigated platforms. This work disclosed that the iSeq platform could be used to evaluate the bacterial profile of the samples to characterize the overall profile. The MiSeq System seems to be better for a detailed analysis of the differences in the microbiota composition. Key points • iSeq platform allows to shorten the sequencing time three times compared to the MiSeq. • iSeq can only be used for an initial and quick microbiome assessment. • MiSeq is better for a detailed analysis of the differences in the microbiota composition.

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“…Tambahkan sebanyak 5µl sampel cDNA kedalam well satu dan dua. Seal dan shake dengan kecepatan 1.000x g selama 1 menit kemudian masukan dalam mesin PCR untuk running (Bhoyar et al, 2021).…”

Section: Library Preparationunclassified

Optimasi dan Evaluasi Whole Genome Sequencing SARS CoV-2 Di Balai Laboratorium Kesehatan dan Pengujian Alat Kesehatan Provinsi Jawa Tengah

Hediningsih¹,

Nurhafid²,

Wahyuni³

et al. 2023

Health Inf J Penelit

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SARS CoV-2 merupakan virus yang menyebar hampir keseluruh Negara di dunia termasuk Indonesia. Whole genome sequencing merupakan metode yang paling tepat digunakan untuk mendeteksi beberapa varian dan mutasi gen dari SARS CoV-2. Tujuan dari penelitian ini yaitu mengetahui dan evaluasi pengerjaan whole genome SARS CoV-2 berdasarkan nilai keberhasilan dan indikator sekuensing di Balai Laboratorium Kesehatan dan Pengujian Alat Kesehatan Provinsi Jawa Tengah. Metode survey digunakan dalam penelitian ini dimana sampel diambil dari beberapa daerah yaitu Jawa tengah, Jawa Barat, Banten dan Jakarta. Analisis keberhasilan didasarkan pada hasil fasta dari analisis Dragon covid lineage. Hasil penelitian menunjukan bahwa persentase keberhasilan dari empat batch pengerjaan menunjukan bahwa dua batch terakhir mendapatkan persentasi lebih tinggi dibandingkan dengan dua batch pertama. Primer artic yang digunakan yaitu versi V3 dan V4 diduga kuat sebagai factor yang mempengaruhi keberhasilan tersebut dimana versi V4 menunjukan performa yang lebih baik dan cocok digunakan dalam pengerjaan whole genome sequencing SARS CoV-2 d Balai Laboratorium Kesehatan dan Pengujian Alat Kesehatan Provinsi Jawa Tengah. Beberapa indikator digunakan sebagai penilaian standar evaluasi yaitu cluster density, passing filter dan >Q30 yang disesuaikan dengan instrument yang digunakan.

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An optimized, amplicon-based approach for sequencing of SARS-CoV-2 from patient samples using COVIDSeq assay on Illumina MiSeq sequencing platforms

Cited by 17 publications

References 4 publications

Simultaneous SARS-CoV-2 Genome Sequencing of 384 Samples on an Illumina MiSeq Instrument through Protocol Optimization

Simultaneous SARS-CoV-2 Genome Sequencing of 384 Samples on an Illumina MiSeq Instrument through Protocol Optimization

Comparison of iSeq and MiSeq as the two platforms for 16S rRNA sequencing in the study of the gut of rat microbiome

Optimasi dan Evaluasi Whole Genome Sequencing SARS CoV-2 Di Balai Laboratorium Kesehatan dan Pengujian Alat Kesehatan Provinsi Jawa Tengah

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