16S rRNA Amplicon Sequencing of Cecal Microbiome

DNA was extracted from 100 mg of cecal contents using the ZymoBIOMICS 96 Magbead DNA Kit from Zymo Research. The sequencing library preparation was performed by following the protocol published by Illumina (document number 15044223, Rev. B). Briefly, the variable regions V3-V4 of the bacterial 16S rRNA gene were targeted by primers designed by Klindworth et al. [38 (link)]. In a two-step protocol, the first round of PCR amplified the target and added Illumina sequencing adapters. Nextera XT set A barcodes were added during the second round of PCR. KAPA HiFi DNA polymerase was used for all amplification reactions. Sequencing was performed on an Illumina MiSeq, using the v3 600-cycle reagent kit to produce 2 × 300 bp paired-end reads. A DNA extraction negative control and mock community DNA (ATCC MSA-1002) were sequenced along with the sample libraries for quality control purposes.

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Rubens J., Kibilds J., Jansons M., Piginka-Vjaceslavova I., Barene I., Daberte I., Liepa L., Malniece A., Rubens A., Starkute V., Zokaityte E., Ruzauskas M., Bartkiene E., Bartkevics V, & Pugajeva I. (2023). Application of Baltic Pine (Pinus sylvestris) Needle Extract as a Gut Microbiota-Modulating Feed Supplement for Domestic Chickens (Gallus gallus). Plants, 12(2), 297.

Publication 2023

ZymoBIOMICS 96 Magbead DNA Kit

16s rrna A dna Bacterial gene Cecal Dna polymerase Library Primers

Corresponding Organization : Institute of Food Safety, Animal Health and Environment “BIOR”

Other organizations : Riga Stradiņš University, Latvia University of Life Sciences and Technologies, Lithuanian University of Health Sciences, University of Latvia

Top 5 similar protocols

Variable analysis

independent variables

DNA extraction method (ZymoBIOMICS 96 Magbead DNA Kit from Zymo Research)
Sequencing library preparation protocol (Illumina document number 15044223, Rev. B)
Primers used to target the variable regions V3-V4 of the bacterial 16S rRNA gene (designed by Klindworth et al.)
Amplification of target and addition of Illumina sequencing adapters (two-step PCR protocol)
Addition of Nextera XT set A barcodes (second round of PCR)
DNA polymerase used for amplification (KAPA HiFi)
Sequencing platform (Illumina MiSeq) and v3 600-cycle reagent kit

dependent variables

Sequencing of 16S rRNA gene variable regions V3-V4

control variables

Sample volume (100 mg of cecal contents)
Sequencing of a DNA extraction negative control
Sequencing of a mock community DNA (ATCC MSA-1002)

controls

Negative control: DNA extraction negative control
Positive control: Mock community DNA (ATCC MSA-1002)

Annotations

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