The V3-V4 hypervariable region of the 16S rRNA gene was amplified by PCR using the following primers: 338F 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′. Subsequent to 16S rRNA library preparation and generation, the library quality was assessed on the [email protected] Fluorometer (Thermo Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system. Then, the libraries’ sequencing was conducted on an Illumina HiSeq platform 2500 (Illumina, SanDiego, CA, USA). The raw data were filtered to exclude low-quality reads. CLC Genomics Workbench 9.5.2 (QIAGEN, Denmark, Germany) was used to merge paired-end reads and generate clean tags, which were then filtered according to Quantitative Insights Into Microbial Ecology (QIIME, version 1.7.0). Operational taxonomic units (OTUs) with ≥97% similarity were determined using the Uparse pipeline (Version 7.0.1001) by clustering all the sequences [21 (link)]. The representative sequence of each OTU was selected, and the taxonomic information was annotated using the RDP classifier [22 (link)] and the GreenGene database at the genus level [23 (link)]. The functional capabilities of gut microbial communities were predicted using the PICRUSt Version 1.0.0 according to the KEGG pathway database.
Illuminahiseq platform 2500
Manufactured by Illumina
Sourced in United States, China, Singapore
The IlluminaHiSeq platform 2500 is a high-throughput DNA sequencing system designed for a variety of genomic applications. It utilizes sequencing-by-synthesis technology to generate large volumes of sequencing data. The core function of the IlluminaHiSeq platform 2500 is to perform massively parallel DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Agilent bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 2000c uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Clc genomics workbench 9, by Qiagen (1 mentions)
Mobio powersoil dna isolation kit, by Qiagen (1 mentions)
3 protocols using illuminahiseq platform 2500
1
16S rRNA Gene Sequencing and Microbial Analysis
2
Isolation and Characterization of Bacterial DNA from Whole Blood
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Isolating genomic DNA from whole blood is classically done using the buffy coat. Bacterial DNA is extracted from peripheral blood leucocytes. Genomic DNA from 200 ul blood samples was isolated using the MoBioPowersoil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA) following the manufacturer’s protocol. The concentration and quality of extracted DNA were assessed photometrically using a NanoDrop® ND-2000c UV–vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The universal primer set 515F (5′-GTG CCA GCM GCC GCG GTA A-3′) and 806R (5′-GGA CTA CNN GGG TAT CTA AT-3′) was used for the amplification of the V4 region of bacterial 16S rRNA gene. After 16S rDNA library preparation and generation, the library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Then, the libraries were sequenced on IlluminaHiSeq platform 2500 and 250 bp paired-end reads were generated at Novogene (Beijing, China).
3
Microbial Community Analysis from Environmental Samples
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gDNA extraction from swabs, MPAs, and tape strips was performed as described above. DNA concentration and quality were assessed photometrically using a NanoDrop ND-2000c UV–vis spectrophotometer (NanoDrop Technologies, Wilmington, DE). The primer set 341F (5′-CCT AYG GGR BGC ASC AG-3′) and 806R (5′-GGA CTA CNN GGG TAT CTA A -3′) was used for the amplification of the V3-V4 region of bacterial 16S ribosomal ribonucleic acid (rRNA) gene. The primer set ITS5 (5′- GGA AGT AAA AGT CGT AAC AAG G-3′) and ITS2 (5′- GCT GCG TTC TTC ATC GAT GC -3′) was used for the amplification of the ITS region of fungal 18S rRNA gene. The libraries were sequenced on IlluminaHiSeq platform 2500, and 250-bp paired-end reads were generated at Novogene AIT (Singapore). Assigned paired-end reads of each sample were merged to raw tags by using Fast Length Adjustment of SHort reads (FLASH) (version 1.2.7), and the merged raw tags were filtered and developed into clean tags according to Quantitative Insights Into Microbial Ecology (QIIME) (version 1.7.0). MUltiple Sequence Comparison by Log-Expectation (MUSCLE) (version 3.8.31) was used to obtain the phylogenetic relationship of all OTU representative sequences. Alpha and beta diversity indices were calculated with QIIME (version 1.7.0).
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