Library preparation was performed on the selected mRNA using NEBNext Ultra RNA Library Prep Kit and Multiplex Oligos for Illumina Sequencing (New England Biolabs). Incubation and PCR steps were carried out by Veriti Thermocycler (Applied Biosystems/Life). During and after the protocol, DNA was purified using Agencourt AMPure XP PCR Purification Beads. Size selection of adapter-ligated DNA was performed immediately prior to final library amplification. This step allowed us to optimize fragment-size distribution for sequencing. Size selection was performed according to NEBNext Ultra Protocol, Version 2.0. Final PCR amplification consisted of 12 cycles. Libraries were then quantified and assessed for quality using an Agilent 2100 Bioanalyzer. Samples were sequenced using the MiSeq Version 2 Reagent Kit on the Illumina MiSeq platform. Samples were prepared according to manufacturer’s protocol (revision B). Each sample was sequenced with a single kit with 151-nucleotide paired-end reads.
Miseq reagent kit version 2
Manufactured by Illumina
Sourced in United States
The MiSeq Reagent Kit version 2 is a lab equipment product from Illumina. It is designed to be used with the MiSeq System for DNA sequencing applications.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (4 mentions)
Nextera xt dna library preparation kit, by Illumina (3 mentions)
Nextera xt index primer, by Illumina (2 mentions)
Qubit double stranded dna high sensitivity assay kit, by Thermo Fisher Scientific (2 mentions)
Agilent hs dna kit, by Agilent Technologies (2 mentions)
Miseq desktop sequencer, by Illumina (2 mentions)
Miseq instrument, by Illumina (2 mentions)
Miseq sequencing system, by Illumina (2 mentions)
Miseq system, by Illumina (2 mentions)
Nebnext ultra rna library prep kit, by Illumina (1 mentions)
Ampure xp pcr purification beads, by Beckman Coulter (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Multiplex oligos for sequencing, by Illumina (1 mentions)
Qubit dna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nextera xt library preparation kit, by Illumina (1 mentions)
Seqsphere, by Ridom (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Qiacube instrument, by Qiagen (1 mentions)
18 protocols using miseq reagent kit version 2
1
mRNA Library Preparation for Illumina Sequencing
2
Whole Genome Sequencing of Bacterial Isolates
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Extracted DNA was quantified using the Qubit double-strandedDNA high-sensitivity assay kit according to the manufacturer’s instructions (Life Technologies Corp., Carlsbad, CA, USA). The Illumina libraries were prepared using the Nextera XT DNA library preparation kit and Nextera XT index primers (Illumina, san Diego, CA, USA). The library fragment size distribution was checked using the Bioanalyzer 2100 with an Agilent HS DNA kit (Agilent Technologies, Santa Clara, CA,USA) and quantified using a Qubit DNA HS assay kit in a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The generated libraries were then sequenced using a MiSeq version 2 reagent kit (Illumina) with 500 and 300 cycles. The paired-end read length of 2 X 250 bp was used for 500 cycles and 2 X 150 bp for 300 cycles on the MiSeq platform (Illumina). The quality metrics of the reads were performed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). The sequences were then assembled using the A5-miseq assembler [22 (link)], and deposited into NCBI under BioProject no. PRJNA679582 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA679582 ). The genome sequence was annotated via the NCBI Prokaryotic Genome Annotation Pipeline [23 (link)].
3
Illumina-based Genome Sequencing and Annotation
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Extracted DNA was quanti ed using the Qubit double-strandedDNA high-sensitivity assay kit according to the manufacturer's instructions (Life Technologies Corp., Carlsbad, CA, USA). The Illumina libraries were prepared using the Nextera XT DNA library preparation kit and Nextera XT index primers (Illumina, san Diego, CA, USA). The library fragment size distribution was checked using the Bioanalyzer 2100 with an Agilent HS DNA kit (Agilent Technologies, Santa Clara, CA,USA) and quanti ed using a Qubit DNA HS assay kit in a Qubit uorometer (Thermo Fisher Scienti c, Waltham, MA, USA). The generated libraries were then sequenced using a MiSeq version 2 reagent kit (Illumina) with 500 and 300 cycles. The pairedend read length of 2 × 250 bp was used for 500 cycles and 2 × 150 bp for 300 cycles on the MiSeq platform (Illumina). The quality metrics of the reads were performed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The sequences were then assembled using the A5-miseq assembler (Coli et al., 2015) , and the genome sequence was annotated via the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2016) (link).
4
Bovine Genome Sequencing and Variant Analysis
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The four enriched multiplex DNA sample libraries were independently sequenced using a MiSeq desktop sequencer (Illumina, Inc.) at The Pirbright Institute. The MiSeq Reagent Kit version 2 (Illumina, Inc.) was used to produce either 2 × 230- or 2 × 250-bp paired-end reads per run. Multiplexed DNA sample libraries were diluted and sequenced at a final concentration of 8 pM. Each sequencing run used a PhiX control spike which was denatured and diluted to 12.5 pM. The final pool of sequenced DNA was comprised of 99% sample library and 1% PhiX. Library preparation, sample loading, and MiSeq preparation steps were carried out as described in the manufacturers’ protocol. Resultant reads were mapped to the cattle genome build UMD_3.1 using the Burrows-Wheeler Aligner (BWA; version 0.7.5a) (Li and Durbin 2009 (link)), and variant sites were identified using SAMtools (version 0.1.18) (Li et al. 2009 (link)) and VarScan (version 2.3.6) (Koboldt et al. 2012 (link)).
5
DNA Extraction and Whole Genome Sequencing Pipeline
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DNA was extracted as previously described (10 (link)). K96243 libraries were prepared using the Nextera XT library preparation kit (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s recommendations. Next-generation sequencing (NGS) was performed on an Illumina MiSeq instrument using MiSeq reagent kit version 2 (Illumina) for a 2- by 250-bp paired-end sequencing run. For all other isolates, NGS was performed on an Illumina HiSeq instrument using HiSeq SBS kit version 4 (Illumina) for a 2- by 100-bp paired-end sequencing run. Raw data were assembled with SKESA (default setting, version 2.3.0) (11 (link)) implemented in SeqSphere+ (version 6.0.92; Ridom GmbH, Münster, Germany).
6
Clonality Analysis of Lymphoid Malignancies
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Genomic DNA (gDNA) was extracted from BM buffy coat samples by using the QIAamp DSP DNA Blood Mini QIAcube Kit (QIAGEN GmbH, Hilden, Germany) and QIAcube instrument (QIAGEN GmbH) or from BM aspirate manually using QIAamp DNA Blood Mini kit (QIAGEN GmbH) according to the manufacturer’s instructions. NGS-based clonality testing was performed using the LymphoTrack IGH FR1 assay kit A-MiSeq (Invivoscribe, Inc. San Diego, CA, USA) and LymphoTrack IGK assay kit A-MiSeq (Invivoscribe, Inc.) according to the manufacturer’s recommendations. Briefly, amplification by PCR was performed using 100 ng of gDNA per each sample, master mixes containing primers designed with barcoded sequence adaptors. After purification and quantification, libraries were sequenced on a MiSeqDx instrument (Illumina, San Diego, CA, USA) using the MiSeq Reagent Kit version 2 (500 cycles) with a length of 2×251 bp for all assays.
7
SARS-CoV-2 Genomic Surveillance in Gwangju City
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Real-time polymerase chain reaction (RT-PCR) was performed using combined nasopharyngeal swab samples and sputum, if available, in the CNUH laboratory, a diagnostic facility for COVID-19 authorized by the KDCA. Whenever possible, whole-genome sequencing (WGS) of specimens from index and secondary cases in the Health and Environmental Research Institute of Gwangju City was conducted to confirm or refute infection. To analyze viral genomic sequences, viral ribonucleic acid (RNA) was extracted using a QIAamp viral RNA Mini Kit (QIAGEN, Hilden, Germany). We prepared WGS libraries using the Illumina COVID Seq Assay kit (Illumina, San Diego, CA, USA) and sequenced them on the MiSeq (Illumina) with 2x150 bp using the MiSeq reagent kit version 2 (Illumina). Additionally, we retrieved data for SARS-CoV-2 genomes uploaded to the GISAID (Global Initiative for Sharing All Influenza Data) database of samples obtained from August to October, 2021 in Gwangju City and analyzed with our samples in the Health and Environmental Research Institute of Gwangju City. For phylogenetic tree analysis, we aligned WGS using the CLC Genomics Workbench (version 21.0.3, QIAGEN, Hilden, Germany), inferred maximum-likelihood phylogenetic trees with Molecular Evolutionary Genetics Analysis (MEGA) (version 10.2.6, The Pennsylvania State Univeristy, PA, USA).
8
Illumina MiSeq 16S Amplicon Sequencing
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Pooled amplicons were sequenced at the Institute for Genomic Medicine at the University of California, San Diego, using the Illumina MiSeq platform. The library concentration was measured using the HiSens Qubit dsDNA HS assay kit (Thermo Fisher Scientific). A total of 6 pM of 16S library combined with 0.9 pM (15%) PhiX sequencing control version 3 was sequenced with 150-bp paired-end (PE) reads on an Illumina MiSeq sequencing system using a MiSeq reagent kit version 2 (300 cycle). Fastq files for reads 1 and 2 and the index read were generated using the BCL-to-FASTQ file converter bcl2fastq version 2.17.1.14 (Illumina, Inc.).
9
Sequencing of 16S rRNA Gene
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Sequencing of the gene encoding 16S rRNA was performed with MiSeq V2 kit according to manufactured procedure (http://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf ). Briefly, the V4 region of the bacterial 16S rDNA was amplified by PCR with forward and reverse primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GTGCCAGCMGCCGCGGTAA-3′ and 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GACTACHVGGGTATCTAATCC-3′, respectively), 5 ng of the DNA from fecal sample, and the TaKaRa Ex Taq HS Kit (TaKaRa Bio, Shiga, Japan). After the PCR products were purified by Agencourt AMPure XP (Beckman Coulter, Inc., CA, USA), the products were amplified using the Nextera Index Kit (Illumina, CA, USA). After the 2nd PCR, amplified products were purified using Agencourt AMPure XP. Library was quantified, normalized and pooled in equimolar amounts according to the manufacturer’s recommendations. Sequencing was conducted using a paired-end to 2 × 150-bp cycle run on an Illumina MiSeq system and MiSeq Reagent Kit version 2 (300 Cycle).
10
Microbiome Profiling from Fecal Samples
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DNA from patient fecal samples was extracted using the QIAamp DNA Stool Mini Kit (catalog No. 51504, Qiagen, Hilden, Germany). For each sample, the V4 region of the 16S rRNA gene was amplified using the dual indexing sequencing strategy, as described previously [14 (link)]. Sequencing was done on the Illumina MiSeq platform using a MiSeq Reagent Kit, version 2 (500 cycles; catalog No. MS102-2003, Illumina Inc, San Diego, CA, USA), according to the manufacturer’s instructions, with modifications found in the Schloss SOP: https://github.com/SchlossLab/MiSeq_WetLab_SOP . The mock community ZymoBIOMICS Microbial Community DNA Standard (catalog No. D6306, Zymo Research, Irvine, CA, USA) was sequenced to monitor sequencing error.
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