AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities.

Area of Science:

• Microbial ecology
• Molecular biology
• Bioinformatics

Background:

• High throughput sequencing (HTSeq) of small ribosomal subunit amplicons offers comprehensive microbial community characterization.
• Existing quality control methods often limit genetic resolution by removing rare sequences or clustering them.
• PCR and sequencing errors introduce noise, complicating accurate microbial community analysis.

Purpose of the Study:

• To propose a simple experimental protocol to enhance the genetic resolution of HTSeq data.
• To effectively remove low-abundance sequences likely resulting from experimental errors.
• To improve the reliability of microbial community census, particularly for rare species.

Main Methods:

• Splitting samples into two halves for independent PCR and sequencing runs.
• Analyzing discordance between the two experimental branches to identify problematic samples.
• Implementing the 'AmpliconDuo filter' to discard sequences not present in both branches.

Main Results:

• The AmpliconDuo filter effectively removes sequences with characteristics of PCR and sequencing errors.
• It retains low-abundance sequences found in both branches, improving the rare biosphere census.
• The filter increases biological resolution by enhancing apparent similarity between similar communities without distorting overall composition.

Conclusions:

• The AmpliconDuo filter is a simple yet effective method for improving the quality and resolution of microbial community data from HTSeq.
• This approach provides a more accurate representation of microbial communities, including rare taxa.
• The filter's impact can be quantitatively explained by a mathematical model.