Abstract
Developments in high-throughput next generation sequencing (NGS) technology have rapidly advanced the understanding of overall microbial ecology as well as occurrence and diversity of specific genes within diverse environments. In the present study, we compared the ability of varying sequencing depths to generate meaningful information about the taxonomic structure and prevalence of antimicrobial resistance genes (ARGs) in the bovine fecal microbial community. Metagenomic sequencing was conducted on eight composite fecal samples originating from four beef cattle feedlots. Metagenomic DNA was sequenced to various depths, D1, D0.5 and D0.25, with average sample read counts of 117, 59 and 26 million, respectively. A comparative analysis of the relative abundance of reads aligning to different phyla and antimicrobial classes indicated that the relative proportions of read assignments remained fairly constant regardless of depth. However, the number of reads being assigned to ARGs as well as to microbial taxa increased significantly with increasing depth. We found a depth of D0.5 was suitable to describe the microbiome and resistome of cattle fecal samples. This study helps define a balance between cost and required sequencing depth to acquire meaningful results.
Highlights
Over the past decade the field of metagenomics has enabled substantial advancement in the knowledge of microbial ecology, evolution, and diversity
Biased nucleic acid extraction procedures are desired to generate DNA for metagenomic shotgun sequencing that accurately reflects the genomic content of the community from which it was derived
All DNA isolation methods from feces can contribute to variation, including differences in cell wall lysis between Gram-positive and Gram-negative bacteria, sensitivity of regions of DNA to inhibitors such as humic acids and the amount of sample extracted[17]
Summary
Rarefaction curves were generated to assess the saturation of samples at each depth for various AMR categories (Fig. 3E,F; Supplementary dataset 3) For both D1 and D0.5, saturation in sequencing was achieved up to the mechanism levels, whereas D0.25 did not reach an asymptote. Previous studies[6,7] indicated a high prevalence of genes within the tetracycline resistance class, with 98% of reads aligning to ribosomal protection proteins represented in TetQ and TetW groups These tetracycline resistance groups were the most prevalent in fecal samples collected from humans[30,31], suggesting their high abundance in both cattle and human populations. Our data demonstrate that D0.5 with ≥50 million reads would be a suitable compromise for sequencing bovine fecal samples and adequately inferring their resistome, considering that no further classes were discovered by the D1 sequencing depth and only a single unique mechanism was discovered as compared to the D0.5 level. Similar pilot studies are recommended for other sample types and matrices prior to undertaking a metagenomics sequencing venture involving a large number of samples
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