16S rDNA Amplicon Sequencing Protocol

The sequencing samples are prepared according to the Illumina 16S rDNA Metagenomic Sequencing Library protocols. The 16S rDNA genes were amplified using 16S rDNA V3-V4 primers (16S rDNA Amplicon PCR Forward Primer: 5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACA GCCTACGGGNGGCWGCAG; 16S rDNA Amplicon PCR Reverse Primer: 5′ GTCTCGTGGGC TCGGAGATGTGTATAAGAGA CAGGACTACHVGGGTATCTAATCC). Input gDNA was amplified with 16S rDNA V3-V4 primers, and a subsequent limited cycle amplification step was performed to add multiplexing indices and Illumina sequencing adapters [39 (link)]. The final products were normalized and pooled using the PicoGreen, and the size of libraries were verified using the TapeStation DNA screentape D1000 (Agilent). And sequencing (2 × 300) was done using the MiSeq™ platform (Illumina) according to the standard protocol.

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Lkhagva E., Chung H.J., Hong J., Tang W.H., Lee S.I., Hong S.T, & Lee S. (2021). The regional diversity of gut microbiome along the GI tract of male C57BL/6 mice. BMC Microbiology, 21, 44.

Publication 2021

16S rDNA Metagenomic Sequencing Library TapeStation DNA screentape D1000 MiSeq™ platform

Genes Library Metagenomic Picogreen Primers Rdna

Corresponding Organization : Kangwon National University Hospital

Other organizations : Jeonbuk National University, Case Western Reserve University, Gyeongsang National University Hospital

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Variable analysis

independent variables

16S rDNA V3-V4 primers (16S rDNA Amplicon PCR Forward Primer: 5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACA GCCTACGGGNGGCWGCAG; 16S rDNA Amplicon PCR Reverse Primer: 5′ GTCTCGTGGGC TCGGAGATGTGTATAAGAGA CAGGACTACHVGGGTATCTAATCC)

dependent variables

Sequencing (2 × 300) using the MiSeq™ platform (Illumina)

control variables

Input gDNA amplification with 16S rDNA V3-V4 primers
Limited cycle amplification step to add multiplexing indices and Illumina sequencing adapters
Normalization and pooling of final products using PicoGreen
Size verification of libraries using TapeStation DNA screentape D1000 (Agilent)

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