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The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons.

Starke IC, Vahjen W, Pieper R, Zentek J - Mol Biol Int (2014)

Bottom Line: A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences.The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters.However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Animal Nutrition, Freie Universität Berlin, Koenigin-Luise-Straße 49, 14195 Berlin, Germany.

ABSTRACT
In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

No MeSH data available.


GC content of sequence reads for two extraction procedures and primer sets. ∗ = significantly different at the respective GC content (P ≤ 0.05).
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Related In: Results  -  Collection


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fig2: GC content of sequence reads for two extraction procedures and primer sets. ∗ = significantly different at the respective GC content (P ≤ 0.05).

Mentions: The GC content of the sequence reads is shown in Figure 2. There were no significant differences for the primer set 968f-1401r for both extraction procedures. However, the primer set 8f-534r led to a significant shift to more GC-rich sequences, when the extraction procedure I was used.


The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons.

Starke IC, Vahjen W, Pieper R, Zentek J - Mol Biol Int (2014)

GC content of sequence reads for two extraction procedures and primer sets. ∗ = significantly different at the respective GC content (P ≤ 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4120916&req=5

fig2: GC content of sequence reads for two extraction procedures and primer sets. ∗ = significantly different at the respective GC content (P ≤ 0.05).
Mentions: The GC content of the sequence reads is shown in Figure 2. There were no significant differences for the primer set 968f-1401r for both extraction procedures. However, the primer set 8f-534r led to a significant shift to more GC-rich sequences, when the extraction procedure I was used.

Bottom Line: A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences.The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters.However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Animal Nutrition, Freie Universität Berlin, Koenigin-Luise-Straße 49, 14195 Berlin, Germany.

ABSTRACT
In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 10(5) sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

No MeSH data available.