Nested PCR Biases in Interpreting Microbial Community Structure in 16S rRNA Gene Sequence Datasets

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• 16S rRNA gene sequencing is crucial for microbial community analysis in human health and environmental studies.
• Standard PCR amplification fails with low DNA concentrations, necessitating alternative methods like nested PCR.
• Potential biases of nested PCR on microbial community structure remain under-investigated.

Purpose of the Study:

• To evaluate the impact of nested PCR on microbial community structure estimation using 16S rRNA gene sequencing.
• To compare nested PCR with standard PCR in vaginal and stool samples with varying microbial diversity.
• To assess the influence of nested PCR cycle numbers on diversity and abundance metrics.

Main Methods:

• Comparison of nested PCR and standard PCR amplification of the 16S rRNA gene from vaginal and stool DNA.
• Analysis of alpha- and beta-diversity metrics and bacterial genus relative abundances.
• Investigation of nested PCR bias using different cycle numbers (10 vs. 20) in the first round.

Main Results:

• Vaginal samples (low diversity) showed no significant difference between nested and standard PCR.
• Stool samples (high diversity) exhibited significant differences in alpha diversity and 13 genera abundances with 20 nested PCR cycles.
• Low-abundance operational taxonomic units (OTUs) contributed significantly to the observed distortions in stool samples.

Conclusions:

• Nested PCR can introduce bias in microbial diversity and community structure estimations, particularly in diverse communities and with increased initial cycles.
• Nested PCR is a viable option when standard PCR is not feasible due to low DNA input.
• Findings from nested PCR, especially concerning rare taxa, require validation using alternative molecular techniques.