Profiling Gut Microbiome Composition

We performed 16S rRNA gene amplicon sequencing on human stool samples to determine microbiota community composition following protocols previously published [41 ]. DNA was extracted from frozen fecal samples with the Qiagen DNeasy PowerSoil DNA extraction kit (ID 12888-100). Amplicon sequencing was performed using custom barcoded primers targeting the V4 region of the 16S rRNA gene [46 (link)], using published protocols [46 (link)–48 (link)]. The sequencing library was diluted to a 5pM concentration and sequenced using an Illumina MiniSeq and a MiniSeq Mid Output Kit (FC420-1004) with paired-end 150bp reads.

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Holmes Z.C., Villa M.M., Durand H.K., Jiang S., Dallow E.P., Petrone B.L., Silverman J.D., Lin P.H, & David L.A. (2022). Microbiota responses to different prebiotics are conserved within individuals and associated with habitual fiber intake. Microbiome, 10, 114.

Publication 2022

DNeasy PowerSoil DNA extraction kit MiniSeq MiniSeq Mid Output Kit

16s rrna Fecal Frozen Gene Human Library Microbiota Primers Rrna gene

Corresponding Organization :

Other organizations : Duke University, Pennsylvania State University

Top 5 similar protocols

Variable analysis

independent variables

DNA extraction method (Qiagen DNeasy PowerSoil DNA extraction kit)

dependent variables

Microbiota community composition

control variables

Frozen fecal samples
Amplicon sequencing targeting the V4 region of the 16S rRNA gene
Illumina MiniSeq sequencing platform with paired-end 150bp reads

controls

None specified
None specified

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