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Assessing the accuracy of quantitative molecular microbial profiling.

O'Sullivan DM, Laver T, Temisak S, Redshaw N, Harris KA, Foy CA, Studholme DJ, Huggett JF - Int J Mol Sci (2014)

Bottom Line: Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq).This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods.We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology, LGC Ltd., Queens Road, Teddington TW11 0LY, UK. denise.osullivan@lgcgroup.com.

ABSTRACT
The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole (meta)genome sequencing (WGS). Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM) to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq). All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR); WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.

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A summary of the four different primer strategies for amplification of variable regions 1, 2, 4, 5 and 6 of the 16S rRNA gene (positioning is based on the E. coli 16S rRNA gene). Highlighted in red are differences in base sequence.
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ijms-15-21476-f002: A summary of the four different primer strategies for amplification of variable regions 1, 2, 4, 5 and 6 of the 16S rRNA gene (positioning is based on the E. coli 16S rRNA gene). Highlighted in red are differences in base sequence.

Mentions: We initially used the MCM to investigate amplicon sequencing by four different primer strategies targeting two different variable regions of the 16S rRNA gene in order to evaluate their impact on quantification (Figure 2). Two strategies (α and β) targeted an amplicon that spanned variable regions 1 and 2 and to compare the measurements using different variable regions we targeted an amplicon to span variable regions 4, 5 and 6 (strategy γ and δ). Both combinations of regions have been investigated previously [20,21,22]. However we redesigned the primers to the respective conserved regions to investigate the impact of different primer strategies on the results. Each experiment performed three replicate runs of the whole protocol including 16S rRNA PCR.


Assessing the accuracy of quantitative molecular microbial profiling.

O'Sullivan DM, Laver T, Temisak S, Redshaw N, Harris KA, Foy CA, Studholme DJ, Huggett JF - Int J Mol Sci (2014)

A summary of the four different primer strategies for amplification of variable regions 1, 2, 4, 5 and 6 of the 16S rRNA gene (positioning is based on the E. coli 16S rRNA gene). Highlighted in red are differences in base sequence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264237&req=5

ijms-15-21476-f002: A summary of the four different primer strategies for amplification of variable regions 1, 2, 4, 5 and 6 of the 16S rRNA gene (positioning is based on the E. coli 16S rRNA gene). Highlighted in red are differences in base sequence.
Mentions: We initially used the MCM to investigate amplicon sequencing by four different primer strategies targeting two different variable regions of the 16S rRNA gene in order to evaluate their impact on quantification (Figure 2). Two strategies (α and β) targeted an amplicon that spanned variable regions 1 and 2 and to compare the measurements using different variable regions we targeted an amplicon to span variable regions 4, 5 and 6 (strategy γ and δ). Both combinations of regions have been investigated previously [20,21,22]. However we redesigned the primers to the respective conserved regions to investigate the impact of different primer strategies on the results. Each experiment performed three replicate runs of the whole protocol including 16S rRNA PCR.

Bottom Line: Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq).This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods.We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology, LGC Ltd., Queens Road, Teddington TW11 0LY, UK. denise.osullivan@lgcgroup.com.

ABSTRACT
The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole (meta)genome sequencing (WGS). Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM) to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq). All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR); WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.

Show MeSH