Impact of sequencing depth on the characterization of the microbiome and resistome

Sci Rep. 2018 Apr 12;8(1):5890. doi: 10.1038/s41598-018-24280-8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anti-Bacterial Agents / pharmacology
  • Archaea / classification
  • Archaea / drug effects
  • Archaea / genetics*
  • Archaea / isolation & purification
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Bacteria / classification
  • Bacteria / drug effects
  • Bacteria / genetics*
  • Bacteria / isolation & purification
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cattle
  • Drug Resistance / genetics*
  • Feces / microbiology
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Fungi / classification
  • Fungi / drug effects
  • Fungi / genetics*
  • Fungi / isolation & purification
  • Gastrointestinal Microbiome / drug effects
  • Gastrointestinal Microbiome / genetics*
  • Gene Expression
  • High-Throughput Nucleotide Sequencing
  • Metagenomics / methods
  • Phylogeny
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • Viruses / classification
  • Viruses / drug effects
  • Viruses / genetics*
  • Viruses / isolation & purification

Substances

  • Anti-Bacterial Agents
  • Archaeal Proteins
  • Bacterial Proteins
  • Fungal Proteins
  • Viral Proteins