Amplicon libraries of the bacterial 16S rRNA gene from water samples and control mock communities were prepared according to the Illumina MiSeq “16S Metagenomic Sequencing Library Preparation” protocol (Illumina, San Diego, CA) and sequenced by an Illumina MiSeq instrument using a MiSeq v3 Reagent Kit according to the “Illumina 16S Metagenomic Sequencing” protocol (Illumina). Sequence data were deposited in the NCBI Sequence Read Archive under BioProject ID: PRJNA391126. Raw sequence data from the forward read were cleaned to remove reads with primer mismatches, missing or low (below 25) quality scores, less than 150 base pairs or greater than 350 base pairs, and more than 6 ambiguous bases. Data from samples with fewer than 100,000 remaining reads were discarded and the sample was resequenced. Sequence reads were further analyzed using Quantitative Insights into Microbial Ecology (QIIME) open source software [26 (link)] to determine the abundance of operational taxonomic units (OTUs) present in each sample. OTUs were defined as a subset of reads sharing ≥97% sequence identity and taxonomic identities were determined with the default classifier used in QIIME (i.e. Ribosomal Database Project).
Miseq 16s metagenomic sequencing library preparation
Manufactured by Illumina
Sourced in United States
The MiSeq 16S Metagenomic Sequencing Library Preparation is a lab equipment product designed for the preparation of metagenomic sequencing libraries targeting the 16S rRNA gene. The core function of this product is to enable the amplification and indexing of the 16S rRNA gene region, which is a commonly used marker for the identification and analysis of microbial communities.
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Lab products found in correlation
Miseq reagent kit v3, by Illumina (4 mentions)
16s metagenomics workflow, by Illumina (1 mentions)
Qubit broad range assay, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
E gel electrophoresis system, by Thermo Fisher Scientific (1 mentions)
Miseq platform, by Illumina (1 mentions)
6 protocols using miseq 16s metagenomic sequencing library preparation
1
Amplicon Sequencing of Bacterial 16S rRNA
2
Bacterial 16S rRNA Profiling of Stool Samples
3
16S Metagenomic Sequencing of Bacterial DNA
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Bacterial DNA was quantified using the Qubit Broad Range assay (ThermoFisher Scientific, Q32853) and 12.5 ngs (5 ng/µl) were used for library preparation following the Illumina MiSeq 16S Metagenomic Sequencing Library Preparation protocol (targeting the V3 and V4 region of the 16S rRNA gene).19 (link) The DNA library was quantified and normalized to 4 nM before pooling, and 5% PhiX (Illumina, FC-110-3001) was added as an internal control. The library was sequenced using the MiSeq Reagent Kit v3 2 × 300 cycles (Illumina, MS-102-3003). Fastq files were generated for further analysis.
4
Faecal Microbiome Profiling by 16S Sequencing
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Microbiota depletion was evaluated by culturing faecal samples in fluid thioglycolate media (Neogen, Lansing, MI, USA) for 3 days in a microbiological incubator at 37 °C and 180 rpm. Alternatively, bacterial load in faecal samples was determined by 16S RNA quantification, as previously described.34 For metagenomic analysis, DNA was extracted from faecal samples using the QIAamp DNA Mini Stool kit (Qiagen, Germantown, MD, USA) and quantified. The 16S rRNA gene amplification and sequencing were performed according to Illumina MiSeq 16S Metagenomic Sequencing Library Preparation protocol at CEFAP/ICB-USP. Amplicons were multiplexed using the Nextera XT Index kit. The quality of libraries was assessed using Bioanalyzer (Agilent, Santa Clara, CA, USA) and quantified using Qubit (Thermo Fisher, Waltham, MA, USA). Sequence analysis was performed using the Qiime package version 1.9.1 and Qiime2.
5
16S rRNA Gene Amplification and Sequencing
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The V3-V4 region of the 16S rRNA gene was amplified by using the following primers: forward primer: 5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG -3’ and reverse primer: 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC -3’. The libraries were constructed according to the protocol (https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html ) for MiSeq 16S Metagenomic Sequencing Library Preparation (Illumina, San Diego, CA, USA) with the Phanta Max Master Mix Kit (Vazyme, Jiangsu, China). As previously described (Ye et al., 2016 (link)), we used three tubes of sterile water as negative controls during the library construction process. No DNA products were detected in the negative controls when evaluated by the E-gel electrophoresis system (Life Technologies). The libraries were then sequenced on a MiSeq platform (Illumina) to generate 2 × 300-bp paired-end reads. Each sample was sequenced for one time.
6
16S rRNA Metagenomic Sequencing Protocol
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Briefly, the V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified from 12.5 ng of stool DNA. The amplicons were then cleaned, sequenced according to the Illumina MiSeq 16S Metagenomic Sequencing Library Preparation protocol [46 ]. The final library was paired-end sequenced at 2 × 300 bp using a MiSeq Reagent Kit v3 on the Illumina MiSeq platform. For bioinformatic analysis, the sequencing data was analyzed using the Illumina 16S metagenomics app (Illumina 16S Metagenimics Pippeline (v1.0.1) [47 ], which performs taxonomic classification of 16S rRNA targeted amplicons using an Illumina-curated version of the GeenGenes taxonomic database. The app provides interactive visualization and raw classification output for per-sample and aggregate analyses. Classification was performed using the Illumina 16 S Metagenomics workflow, which is also available in the MiSeq Reporter software. The algorithm uses a high-performance implementation of the Ribosomal Database Project (RDP) Classifier described in Wang et al., 2007 [48 (link)].
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