Enhancing the Resolution of Rumen Microbial Classification from Metatranscriptomic Data Using Kraken and Mothur

Area of Science:

• Microbial ecology
• Bioinformatics
• Rumen microbiome

Background:

• Next-generation sequencing and bioinformatics tools advance gut microbiome research.
• Limited evaluation of these tools for the rumen microbiome exists, as most studies focus on human data.

Purpose of the Study:

• To compare Kraken (mRNA-based) and Mothur (16S rRNA-based) for taxonomic profiling of the rumen microbiome in cattle.
• To assess the accuracy and comprehensiveness of each method for bacterial and archaeal identification.

Main Methods:

• Total RNA sequencing of rumen fluid from 12 cattle with varying feed conversion ratios (FCR).
• Analysis using Kraken and an in-house Mothur pipeline.
• Comparison of taxonomic profiles at phylum, genus, and species levels for bacteria and archaea.

Main Results:

• Both methods showed similar phyla distribution (Bacteroidetes, Firmicutes, Proteobacteria dominant).
• Kraken identified significantly more bacterial genera and species than Mothur.
• Archaea identification showed similarities for Methanomassiliicoccaceae, but differences in specific taxa (R4-41B, Methanococcaceae) were noted.
• Limited reference genomes for the rumen microbiome impact identification accuracy.

Conclusions:

• Kraken offers deeper taxonomic resolution, particularly at the species level, for rumen bacteria.
• Mothur and Kraken provide complementary insights into rumen microbial communities.
• Developing combined pipelines is crucial for comprehensive rumen microbiome classification due to current reference limitations.