Gut Microbiome Profiling via 16S rRNA Sequencing

DNA was extracted from approximately 0.25-g aliquots of thawed stool samples by using the DNeasy PowerSoil extraction kit (Qiagen, Hilden, Germany) and the automated QIAcube platform (inhibitor removal technology [IRT] protocol). The V5-V6 hypervariable regions of the 16S rRNA gene were amplified using the BSF784/1064R primer set (40 (link)). Illumina adapters (Illumina Inc., San Diego, CA, USA) and barcodes were appended to amplicons using the dual-index method by the University of Minnesota Genomics Center (UMGC) (41 (link)), and paired-end sequencing was done on the Illumina MiSeq platform at a read length of 300 nucleotides (nt).

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Staley C., Kaiser T., Vaughn B.P., Graiziger C., Hamilton M.J., Kabage A.J., Khoruts A, & Sadowsky M.J. (2019). Durable Long-Term Bacterial Engraftment following Encapsulated Fecal Microbiota Transplantation To Treat Clostridium difficile Infection. mBio, 10(4), e01586-19.

Publication 2019

DNeasy PowerSoil extraction kit Illumina adapters MiSeq platform

Nucleotides Primer Rrna gene Stool

Corresponding Organization :

Other organizations : University of Minnesota

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Variable analysis

independent variables

DNA extraction method (DNeasy PowerSoil extraction kit)
Automated extraction platform (QIAcube platform)
Primer set used for 16S rRNA gene amplification (BSF784/1064R)

dependent variables

Sequencing of the V5-V6 hypervariable regions of the 16S rRNA gene
Paired-end sequencing on the Illumina MiSeq platform

control variables

Aliquot size of thawed stool samples (approximately 0.25 g)
Read length of sequencing (300 nucleotides)

controls

No positive or negative controls were explicitly mentioned in the protocol.

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