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

  1. Justin P. Shaffer
  2. Clarisse Marotz
  3. Pedro Belda-Ferre
  4. Cameron Martino
  5. Stephen Wandro
  6. Mehrbod Estaki
  7. Rodolfo A. Salido
  8. Carolina S. Carpenter
  9. Livia S. Zaramela
  10. Jeremiah J. Minich
  11. MacKenzie Bryant
  12. Karenina Sanders
  13. Serena Fraraccio
  14. Gail Ackermann
  15. Gregory Humphrey
  16. Austin D. Swafford
  17. Sandrine Miller-Montgomery
  18. Rob Knight
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Abstract

One goal among 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 we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we 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.

Accession numbers

Raw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://github.com/justinshaffer/Extraction_test_MagMAX">github.com/justinshaffer/Extraction_test_MagMAX</ext-link>).

Methods summary

To allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

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