Research Summary - 3
Target-enriched sequencing enables genomic characterization of Mannheimia haemolytica within diverse microbial populations
| Date/Time: |
9/13/2025
11:15
|
| Author: |
Enrique Doster |
| Clinic: |
VERO |
| City, State, ZIP: |
Canyon, TX 79015 |
E. Doster, DVM, PhD, BS
1
;
L.J. Pinnell, PhD, MS, BS
1
;
Noyes N.R., DVM, PhD, MA, BA
2
;
Valeris-Chacin R., DVM, PhD, MS, BS
1
;
Crosby W.B., DVM, PhD, BS
3
;
Clawson M.L., PhD, BS
4
;
Woolums A.R., DVM, PhD, DACVM, BS
3
;
Morley P.S., DVM, PhD, DACVIM, BS
1
;
1Veterinary Education, Research, and Outreach Program, Texas A&M University, Canyon, TX
2Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, MN
3Department of Pathobiology and Population Medicine, Mississippi State University, Starkville, MS
4USDA, Agriculture Research Service - US Meat Animal Research Center, Clay Center, NE
Introduction:
Mannheimia haemolytica (Mh) is a key pathogen associated with Bovine Respiratory Disease (BRD). Previously, investigation of genetic variation that may affect microbial ecology and disease epidemiology was reliant upon the ability to culture Mh and perform whole genome sequencing. This study describes a comprehensive target-enriched shotgun sequencing approach that provides a culture-independent method for detection and sequencing of Mh in metagenomic microbial communities, allowing variant or strain-level investigations of Mh communities.
Materials and methods:
We created a customized probe set specific for Mh using the Agilent SureSelect system for target enriched (TE) shotgun sequencing. Specialized methods for TE library preparation were developed and tested. This system was used to perform TE sequencing on DNA recovered from nasal swab samples collected from a single group of 39 feedlot cattle. These samples were previously characterized using culture, qPCR for Mh, and 16S rRNA amplicon sequencing. This set was purposefully selected based on stratification of the Mannheimia spp. relative abundance determined via 16S sequencing (>30%, 10-30%, 1-10%, 0.1-1%, and 0%). To address challenges for variant-level classification of sequencing reads, we refined the bioinformatic process by clustering all publicly available Mh genomes (n=2,106) by sequence similarity, creating groups of "genomic sequence variants” (GSVs). This data-driven approach is designed to enhance variant-level resolution of short-read sequencing classification, leveraging data from all published Mh genome sequences. We developed the VARIANT++ pipeline for high-throughput analysis of these sequencing data, incorporating QC trimming, read merging and deduplication, host DNA removal, and taxonomic classification of bacterial species and GSVs for Mh.
Results:
Customized TE sequencing successfully enriched sequences in these metagenomic samples, producing an average of 90% of reads per sample (range 50-93%) that were classified to Mh at the species level. TE sequencing had greater sensitivity for Mh detection than culture, qPCR, and 16S amplicon sequencing, as demonstrated by detection of Mh culture-negative samples (6/6) and those with zero reads classified as Mannheimia spp. by 16S sequencing (4/4). Further taxonomic classification identified 6 unique GSVs within the sample set, suggesting that multiple genetic variants of Mh were present within this single group of cattle. Preliminary investigation of other samples suggests similar complexity of Mh variants are found in other cattle populations, with greater variations in low-abundance GSVs.
Significance:
This novel TE metagenomic approach enables variant-level characterization of Mh with increased sensitivity and specificity compared to shotgun sequencing, bacterial culture, qPCR, and 16S amplicon sequencing. It enables novel investigations into the dynamics of bovine respiratory microbial communities. Further research is needed to investigate the significant identifying diverse GSVs in groups of cattle, and potential associations with BRD epidemiology.
