The effects of variable sample biomass on comparative metagenomics

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Longitudinal studies are crucial for understanding microbial community dynamics.
• Shotgun metagenomics is a key tool for functional analysis of microbial communities.
• The impact of variable biomass on metagenomic analysis is not well understood.

Purpose of the Study:

• To investigate the effects of DNA input quantity and polymerase chain reaction (PCR) amplification on comparative metagenomic analysis.
• To assess the influence of these factors on microbial community structure and downstream analysis.
• To provide recommendations for handling low biomass or heterogeneous samples in metagenomic surveys.

Main Methods:

• Utilized dilutions of a single complex microbial community template from Arabidopsis thaliana.
• Modified the Illumina Nextera kit for large-insert paired-end library preparation (680 bp).
• Employed a range of DNA input from 50 pg to 50 ng.
• Performed assembly-based metagenomic analysis.

Main Results:

• DNA input level significantly impacts microbial community structure due to biased amplification of low-GC genomic regions.
• These biases were largely overcome by variations between biological replicates in the tested system.
• The study identified specific impacts of library amplification on metagenomic data.

Conclusions:

• Library amplification can introduce biases in metagenomic analysis, particularly affecting community structure.
• Recommendations are provided for quality filtering and de-replication to mitigate these effects.
• A framework is presented for addressing biomass heterogeneity in longitudinal metagenomic studies.