# Genome-resolved metagenomics uncovers diversity and functional landscapes of the gastrointestinal epithelium-associated microbiome in cattle

## TL;DR

A new sequencing method reveals the diverse and functionally important microbes in cattle gut epithelium, uncovering new species and metabolic pathways.

## Contribution

Dilute-MetaSeq, a novel sequencing workflow, enables high-efficiency microbial analysis of the cattle gastrointestinal epithelium microbiome.

## Key findings

- Dilute-MetaSeq reduces host DNA interference by 52.4-fold and achieves >90% microbial sequencing efficiency.
- The MGA-GE resource includes 1,907 prokaryotic and 5,603 viral genomes, with most being taxonomically unclassified.

## Abstract

The ruminant gastrointestinal epithelium harbors a diverse and functionally critical remains poorly characterized microbial community due to persistent host-derived DNA contamination in metagenomic studies.

We develop Dilute-MetaSeq (dilution-based metagenomic sequencing), a novel, metagenomic workflow integrating gradient dilution with multiple displacement amplification. Dilute-MetaSeq reduces host DNA interference by 52.4-fold and achieves > 90% microbial sequencing efficiency to assess gastrointestinal epithelium-associated microbiome. This enables the construction of the microbial genome atlas of gastrointestinal epithelium (MGA-GE). This comprehensive resource, comprising 1,907 nonredundant prokaryotic and 5,603 viral genomes, reveals extraordinary microbial diversity and novelty, with 41.4% of prokaryotic and 99.9% of viral genomes representing taxonomically unclassified lineages. Spatial profiling identifies the rumen and reticulum as a biodiversity hotspot dominated by epithelium-adapted Butyrivibrio and methylotrophic Methanomassiliicoccales, while functional annotation uncovers 1,200 biosynthetic gene clusters (primarily RiPPs and NRPSs) and 1,212 viral auxiliary metabolic genes linked to host metabolism modulation. Pangenome analysis of 987 strains, including a novel Butyrivibrio clade with reduced genome sizes, elevated GC content, and butyrate synthesis from amino acid-derived substrates (e.g., glutarate, lysine), highlights metabolic adaptations to the nutrient-scarce epithelial niche compared to digesta-associated microbes.

Collectively, the MGA-GE provides transformative insights into host-microbe-virus interactions and establishes a foundation for developing microbiome-based intervention strategies to enhance ruminant health, agricultural productivity, and bioactive discovery.

The online version contains supplementary material available at 10.1186/s13059-026-03960-z.