16S rRNA Gene Amplification and Sequencing

The V3 and V4 regions of 16S rRNA gene were amplified using primers (341F 5′-CCTACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACNNGGGTATCTAAT-3′). PCR products were purified with GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Massachusetts, USA). Sequencing libraries were generated using NEB Next Ultra DNA Library Prep Kit for Illumina (NEB, Ipswich, USA) and library quality was assessed on the Qubit@ 2.0 Flourometer (Thermo Fisher Scientific, Massachusetts, USA) and Agilent Bioanalyzer 2100 system. The amplicon library was sequenced on an Illumina HiSeq 2500 platform, generating 250 bp paired-end reads. Paired-end reads were merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/)^{39 (link)} and assigned to each sample according to the unique barcodes. The sequence reads were quality filtered using QIIME (V1.7.0)^{40 (link)} and compared with the reference database (Gold database, http://drive5.com/uchime/uchime_download.html) using UCHIME algorithm^{41 (link)} to detect and remove chimeric sequences^{42 (link)}. The reads were clustered using Uparse software (Uparse v7.0.1001)^{43 (link)} and OTUs (Operational Taxonomic Units) generated based on 97% sequence homology and OTU representative sequences were then classified taxonomically using the QIIME-based wrapper of the Ribosomal Database Project (Version 2.2)^{44 (link)}.

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Lindroth K.M., Dicksved J., Pelve E., Båverud V, & Müller C.E. (2021). Faecal bacterial composition in horses with and without free faecal liquid: a case control study. Scientific Reports, 11, 4745.

Publication 2021

GeneJET Gel Extraction Kit NEB Next Ultra DNA Library Prep Kit for Illumina Qubit@ 2.0 Flourometer Agilent Bioanalyzer 2100 system HiSeq 2500 platform

16s rrna Chimeric Gene Gold Library Primers Ribosomal

Corresponding Organization : Swedish University of Agricultural Sciences

Other organizations : Swedish Veterinary Agency

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Variable analysis

independent variables

Primers (341F 5′-CCTACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACNNGGGTATCTAAT-3′) used to amplify the V3 and V4 regions of 16S rRNA gene

dependent variables

Sequencing data generated from the amplified 16S rRNA gene regions

control variables

Use of the GeneJET Gel Extraction Kit to purify the PCR products
Use of the NEB Next Ultra DNA Library Prep Kit for Illumina to generate sequencing libraries
Use of the Qubit@ 2.0 Flourometer and Agilent Bioanalyzer 2100 system to assess library quality
Use of the Illumina HiSeq 2500 platform to sequence the amplicon library
Use of FLASH (V1.2.7) to merge the paired-end reads
Use of QIIME (V1.7.0) to quality filter the sequence reads
Use of the UCHIME algorithm to detect and remove chimeric sequences
Use of the Uparse software (Uparse v7.0.1001) to cluster the reads and generate OTUs
Use of the QIIME-based wrapper of the Ribosomal Database Project (Version 2.2) to taxonomically classify the OTU representative sequences

controls

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

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