20 nM of pooled 16S rRNA gene library and 20 nM of PhiX control v3 (Illumina) were mixed with 0.2 N of fresh NaOH and HT1 buffer (Illumina) to produce the final concentration at 7.8 pM. The resulting library was mixed with the PhiX control v3 (10%, v/v, Illumina) and 600 µl loaded on a MiSeq® v2 Reagent cartridge (500 cycle, Illumina) for sequencing.
Phix control v3
Manufactured by Illumina
Sourced in United States, Canada
PhiX Control v3 is a prepared DNA library that serves as a control sample for next-generation sequencing (NGS) experiments. It provides a known, high-quality DNA sequence that can be used to assess the performance and quality of the sequencing run.
Automatically generated - may contain errors
Lab products found in correlation
Miseq reagent kit v3, by Illumina (14 mentions)
Miseq platform, by Illumina (10 mentions)
Miseq reagent kit v2, by Illumina (10 mentions)
Miseq system, by Illumina (9 mentions)
Nextseq 500, by Illumina (8 mentions)
Miseq sequencer, by Illumina (8 mentions)
Ht1 buffer, by Illumina (7 mentions)
Ampure xp bead, by Beckman Coulter (7 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (7 mentions)
Miseq instrument, by Illumina (6 mentions)
Nextera xt index kit, by Illumina (5 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Nextera xt index kit v2, by Illumina (4 mentions)
Miseq reagent kit, by Illumina (4 mentions)
Qiaquick pcr purification kit, by Qiagen (4 mentions)
Kapa hifi hotstart readymix, by Roche (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Miseq cartridge, by Illumina (3 mentions)
Quantifluor dsdna system, by Promega (3 mentions)
94 protocols using phix control v3
1
16S rRNA Gene Sequencing on MiSeq
2
16S rRNA Sequencing on MiSeq
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A 20 nM pool of the 16S rRNA library and 20 nM PhiX control V3 (Illumina) were mixed with 0.2 N of fresh NaOH and HT1 buffer (Illumina) to produce the final concentration of 6 pM. The resulting library was mixed with the PhiX control V3 (5%, vol/vol, Illumina) and 600 µL loaded on a MiSeq v2 (500 cycle, 2 × 250 bp) reagent cartridge for sequencing. The 16S rRNA amplicon sequences are available at the Sequence Read Archive of the National Center for Biotechnology Information (https: / / dataview .ncbi .nlm .nih .gov/ object/ PRJNA591223 ?reviewer = ktedeigveld5k4n89ljtkr3f0l) under SUB6594737.
3
Dilution and Mixing of Sequencing Library
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Each of the 20 nM of pooled library and 20 nM of PhiX control v3 (Illumina) were diluted with 0.2 N fresh NaOH and HT1 buffer (Illumina) to produce the final concentration at 7.8 pM. The diluted library was mixed with the PhiX control v3 (10%, vol/vol, Illumina) and
4
Whole-Genome Sequencing of 1000 Genomes Samples
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DNA extracted from LCLs was ordered from the Coriell Institute for Medical Research for each of the 3,202 1kGP samples. Whole-genome sequencing (WGS) libraries were prepared using the TruSeq DNA PCR-Free High Throughput Library Prep Kit in accordance with the manufacturer’s instructions. Briefly, 1ug of DNA was sheared using a Covaris LE220 sonicator (adaptive focused acoustics). DNA fragments underwent bead-based size selection (SPRIselect, Beckman Coulter) and were subsequently end-repaired, adenylated, and ligated to Illumina sequencing adapters (IDT for Illumina – TruSeq DNA UD Indexes (Illumina)). Final libraries were evaluated using fluorescent-based assays including measuring concentration with Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies), qPCR with the Universal KAPA Library Quantification Kit and Fragment Analyzer (HS NGS Fragment Kit, Agilent) or BioAnalyzer (Agilent 2100). Libraries were sequenced on an Illumina NovaSeq 6000 system using 2 x 150bp cycles (NovaSeq 6000 S4 Reagent kit; NovaSeq Xp Kit; PhiX v3 Control (Illumina)).
5
MiSeq Sequencing of Amplicon Library
6
Illumina Sequencing Library Preparation
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After isolation, 200 ng of each isolate was used for sequencing on the MiSeq and iSeq (Illumina, CA). Both sequencing libraries were prepared on an epMotion 5075 TC liquid handler (Eppendorf, Hamburg, DE) using the ILMN DNA LP (M) Tagmentation 96 IPB kit protocol as described by the manufacturer. The pooled libraries were spiked with 1% v/v PhiX Control V3 (Illumina, San Diego, CA) and were diluted to a final loading concentration of 10 pM and 100 pM for the MiSeq and iSeq, respectively. The diluted libraries (600 µL and 20 µL) were loaded onto a MiSeq Reagent Nano Kit v2 (500-cycles) and iSeq 100 i1 Reagent v2 (300-cycle). FASTQ files generated were used for sequence validation, comparison, and evaluation.
7
Illumina MiSeq Sequencing Library Preparation
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The pool was denatured (NaOH 0.1N) and diluted to 7 pM. PhiX Control v3 (Illumina, San Diego, USA) was added to the pool at 4.5% of the final concentration. From this mixture, 600 μl were loaded onto the Illumina MiSeq cartridge according to the manufacturer’s instructions using the MiSeq Reagent Kit v3 (2x300 bp paired-end reads, 15 Gb output). FastQ files were generated at the end of the run (MiSeq Reporter software, Illumina, USA) for quality control. The quality of the run was checked internally using PhiX Control and then each paired-end sequence was assigned to its sample of origin using the multiplexing index tag. Raw read sequences were deposited at the Sequence Read Archive under the accession numbers SAMN09070427to SAMN09070506.
8
MiSeq Sequencing of Amplicons
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Sequencing of the amplicons was conducted at University of New South Wales (UNSW) using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) with a paired-end 300 base pair sequencing protocol. A pooled library (20 nM) and a PhiX control v3 (20 nM) (Illumina) were mixed with 0.2 N fresh NaOH and HT1 buffer (Illumina) to produce the final concentration at 12 pM each. The resulting library was mixed with the PhiX control v3 (5%, v/v) (Illumina) and 600 μl loaded on a MiSeq1 v2 (500 cycles) Reagent cartridge for sequencing. All sequencing procedures were monitored through the Illumina BaseSpace® website.
9
Amplicon library preparation for Illumina sequencing
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Sequencing libraries were generated by three rounds of PCR, according to previously published protocols9 (link). After primary PCR, a 5′ linker sequence was added during nested PCR. Nested PCR products were subject to another PCR round with primers binding to the linker sequences and carrying Illumina sequence adapters plus an eight nucleotide long sample-specific molecular index to permit pooling of amplicons for sequencing and later de-multiplexing. The final sequence library was purified with NucleoMag beads. Sequencing was performed on an Illumina MiSeq platform in paired-end mode (2 × 250 bp) using Illumina MiSeq reagent kit v2 (500-cycles) together with Enterobacteria phage PhiX control (Illumina, PhiXControl v3).
10
SARS-CoV-2 Target Capture Enrichment Protocol
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Target capture‐based enrichment was used for SARS‐CoV‐2 library preparation. The target capture custom probes were designed to cover the entire sequence of the reference strain SARS‐CoV‐2 (Wuhan‐Hu‐1; NC_045512). Overall, 745 biotinylated probes were designed. Each probe was 120 bp with ×3 tiling, and the total probe size was 29.9 kb (Celemics). TruSeq RNA Library Prep for Enrichment (Illumina) was used for library preparation. The reaction was performed with 10–100 ng of total RNA extracted from the saliva. All RNA samples were reverse‐transcribed into cDNA, and adapters were ligated to the ends using the dual indices set (Illumina). The adapter‐ligated libraries and amplified libraries were purified using AMPure XP beads (Beckman Coulter). The library quality and concentration were determined using TapeStation 4200 and D1000 ScreenTape (Agilent Technologies). The libraries were quantified using the KAPA Library Quantification kit (KAPA Biosystems) on a QuantStudio 6 Flex Real‐time PCR system (Thermo Fisher Scientific). The final library concentration was 4–14 pM, with 1–10% PhiX control v3 (Illumina). NGS of the enriched samples was performed on a MiSeq and Nextseq. 500 benchtop platform (Illumina) using MiSeq Reagent v2 Kit 300 Cycle (2 × 150 bp) and Nextseq. 500/550 Mid Output Kit v2.5 (Illumina).
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