A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

Biotechniques. 2021 Mar;70(3):149-159. doi: 10.2144/btn-2020-0153. Epub 2021 Jan 29.


One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

Keywords: 16S rRNA; DNA extraction; RNA extraction; high-throughput sequencing; limit of detection; microbial community; microbiome; shotgun metagenomics; well-to-well contamination.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biodiversity
  • Cats
  • Chemical Fractionation / methods
  • DNA, Viral / isolation & purification*
  • Feces / microbiology
  • Feces / virology
  • Female
  • Fermented Foods / microbiology
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Limit of Detection
  • Male
  • Metagenomics / methods
  • Mice
  • Microbiota / genetics*
  • RNA, Ribosomal, 16S / isolation & purification*
  • SARS-CoV-2 / genetics*
  • Saliva / microbiology
  • Saliva / virology
  • Skin / microbiology
  • Skin / virology


  • DNA, Viral
  • RNA, Ribosomal, 16S