Library preparation was performed using a two-step PCR amplification, first using Q5 High-Fidelity DNA Polymerase (New England Biolabs Limited, Hitchin, UK) and 16S universal primers targeting the V3–V4 region of the bacterial 16S rRNA gene as outlined above. The resulting single amplicon target of approximately ~460 bp was amplified using a second limited cycle PCR adding Illumina sequencing adaptors and dual-index barcodes. Samples were sequenced by Illumina MiSeq using paired 300 bp reads and MiSeq v3 reagents.
Miseq v3 reagents
Manufactured by Illumina
Sourced in United States
The MiSeq v3 reagents are a set of reagents designed for use with the Illumina MiSeq sequencing platform. The core function of these reagents is to enable DNA sequencing on the MiSeq instrument.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt index kit, by Illumina (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Qubit 2, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
High sensitivity dna kit, by Agilent Technologies (1 mentions)
16s metagenomic sequencing library preparation protocol, by Illumina (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
Miseq platform, by Illumina (1 mentions)
Maxima h minus double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sars cov 2 respiratory panel, by Illumina (1 mentions)
Agilent 2200 tapestation instrument, by Agilent Technologies (1 mentions)
Invitrogen dnase 1 amplification grade, by Thermo Fisher Scientific (1 mentions)
Miseq system, by Illumina (1 mentions)
7 protocols using miseq v3 reagents
1
16S rRNA Gene Amplicon Sequencing
2
Illumina MiSeq 16S rRNA Sequencing Workflow
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Illumina MiSeq paired-end sequencing of the hypervariable V3-V4 regions of the 16S rRNA was performed at Bioengineering Lab. Co., Ltd., Kanagawa, Japan. A two-step, tailed PCR approach was used according to the protocol for 16S metagenomic sequencing library preparation (Illumina, San Diego, CA, USA). Both the V3 and V4 regions of the 16S ribosomal RNA were amplified with primers containing the Illumina overhang adaptor (Forward primer 5′ ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT CCT ACG GGN GGC WGC AG; Reverse primer 5′ GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TGA CTA CHV GGG TAT CTA ATC C). Index PCR was performed with Index 1 and Index 2 Primers from the Nextera XT Index Kit (Illumina), using 2 μL of amplicon derived from the previous PCR. The indexed libraries were cleaned and analysed with Bioanalyzer, using a high sensitivity DNA kit (Agilent Technologies, Wilmington, DE). The prepared libraries were used for paired-end sequencing using MiSeq v3 reagents and 2 × 300-bp reads on the MiSeq (Illumina).
3
Amplification and Sequencing of 16S rRNA
4
16S rRNA Gene Amplicon Sequencing
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The microbial DNA was extracted using a QIAamp® DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For next-generation sequencing, microbial DNA primers targeted the 16S V3 and V4 region were used (PCR conditions: 3 min 95 °C; 25 cycles: 30 s 95 °C, 30 s 55 °C, 30 s 72 °C; 5 min 72 °C; and held 4 °C; Mastercycler Gradient, Eppendorf AG, Hamburg, Germany). Subsequent DNA quality was assessed by applying a Bioanalyzer DNA 1000 chip. For DNA purification, Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) were used according to the manufacturer’s protocol. The Nextera XT Index Kit (Illumina, San Diego, CA, USA) was employed for dual indexing of different DNA samples (index PCR conditions: 3 min 95 °C; 8 cycles: 30 s 95 °C, 30 s 55 °C, 30 s 72 °C; 5 min 72 °C; and held 4 °C). After DNA purification and quality control, the DNA was evaluated using a Qubit® dsDNA BR Assay Kit and Qubit® Fluorometer (Thermofisher Scientific, Waltham, MA, USA) for library quantification and normalization. The different DNA samples were pooled (final concentration of 4 pM) and spiked with phiX control (4 pM). For sequencing analysis, MiSeq v3 reagents (Illumina, San Diego, CA, USA) were applied.
5
Amplicon Sequencing on Illumina MiSeq
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Amplicons, approximately 390 bp length were subjected to index PCR creating 460 bp libraries. These were pooled according to Illumina protocols [19 ] and sequenced on a MiSeq instrument using paired 300 bp reads and MiSeq v3 reagents (MS-102-3003, Illumina). The indices for each sample are provided in S1 Table .
6
Illumina 16S Metagenomic Library Preparation
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We followed the Illumina 16S Metagenomics Library Preparation manual (Illumina, Inc., San Diego, CA) according to the manufacturer’s protocol. Briefly, we used previously designed primers to isolate the hyper-variable V3 and V4 region of the 16S rRNA amplicon (Klindworth et al., 2013 (link)), the samples were barcoded using Nextera indexes, and an amplicon of approximately 460 bp was generated. The libraries were pooled and sequenced on the Illumina MiSeq platform, using paired-end 300 bp reads and Illumina MiSeq v3 reagents. The end of each read was overlapped to generate high quality, full-length reads of the V3 and V4 regions.
7
SARS-CoV-2 Viral Genome Sequencing from Nasopharyngeal Swabs
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RNA extracted from nasopharyngeal swabs were treated with Invitrogen DNase I, Amplification Grade (ThermoFisher Scientific, Waltham, MA, USA). The concentration and the quality of isolated RNA samples were measured with the Qubit 2.0 (ThermoFisher Scientific) and Agilent 2200 Tape Station Instrument, respectively. Total RNA was reverse transcribed into cDNA with Maxima H Minus Double-Stranded cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA). The resulting dsDNA was purified using 1.8× Agencourt AMPure XP Beads (Beckman Coulter, Woerden, Netherlands) and quantified with Qubit 2.0 (ThermoFisher Scientific).
Sequencing-ready libraries were prepared using the SARS-Cov-2 Respiratory panel with the DNA Prep with Enrichment and IDT for Nextera DNA UD Indexes from Illumina (San Diego, CA, USA) according to the manufacturer's instructions. Viral enriched libraries were sequenced on the MiSeq System at 2 × 75 bp read length using MiSeq v3 reagents (Illumina).
Sequencing-ready libraries were prepared using the SARS-Cov-2 Respiratory panel with the DNA Prep with Enrichment and IDT for Nextera DNA UD Indexes from Illumina (San Diego, CA, USA) according to the manufacturer's instructions. Viral enriched libraries were sequenced on the MiSeq System at 2 × 75 bp read length using MiSeq v3 reagents (Illumina).
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