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Library Preparation and Sequencing Platform Introduce Bias in Metagenomic-Based Characterizations of Microbiomes

View ORCID ProfileCasper S. Poulsen, Claus T. Ekstrøm, Frank M. Aarestrup, View ORCID ProfileSünje J. Pamp
doi: https://doi.org/10.1101/592154
Casper S. Poulsen
aResearch Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark
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Claus T. Ekstrøm
bSection of Biostatistics, Department of Public Health, University of Copenhagen, Copenhagen, Denmark
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Frank M. Aarestrup
aResearch Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark
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Sünje J. Pamp
aResearch Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark
cNovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark
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  • For correspondence: sjpa@dtu.dk
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Abstract

Metagenomics is increasingly used to describe microbial communities in biological specimens. Ideally, the steps involved in the processing of the biological specimens should not change the microbiome composition in a way that it could lead to false interpretations of inferred microbial community composition. Common steps in sample preparation include sample collection, storage, DNA isolation, library preparation, and DNA sequencing. Here we assess the effect of three library preparation kits and two DNA sequencing platforms. Of the library preparation kits, one involved a polymerase chain reaction (PCR) step (Nextera), and two were PCR-free (NEXTflex and KAPA). We sequenced the libraries on Illumina HiSeq and NextSeq platforms. As example microbiomes, we assessed two pig fecal samples and two sewage samples of which aliquots were stored at different storage conditions (immediate processing and storage at −80°C). All DNA isolations were performed in duplicate, totaling 80 samples excluding controls. We found that both library preparation and sequencing platform had systematic effects on the inferred microbial community composition. The different sequencing platforms introduced more variation than library preparation and freezing the samples. The results highlight that all sample processing steps need to be considered when comparing studies. Standardization of sample processing is key to generate comparable data within a study, and comparisons of differently generated data, such as in a meta-analysis, should be performed cautiously.

Importance Previous research has reported effects of sample storage conditions and DNA isolation procedures on metagenomics-based microbiome composition; however, the effect of library preparation and DNA sequencing in metagenomics has not been thoroughly assessed. Here, we provide evidence that library preparation and sequencing platform introduce systematic biases in the metagenomic-based characterization of microbial communities. These findings suggest that library preparation and sequencing are important parameters to keep consistent when aiming to detect small changes in microbiome community structure. Overall, we recommend that all samples in a microbiome study are processed in the same way to limit unwanted variations that could lead to false conclusions. Furthermore, if we are to obtain a more holistic insight from microbiome data generated around the world, we will need to provide more detailed sample metadata, including information about the different sample processing procedures, together with the DNA sequencing data at the public repositories.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted January 11, 2022.
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Library Preparation and Sequencing Platform Introduce Bias in Metagenomic-Based Characterizations of Microbiomes
Casper S. Poulsen, Claus T. Ekstrøm, Frank M. Aarestrup, Sünje J. Pamp
bioRxiv 592154; doi: https://doi.org/10.1101/592154
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Library Preparation and Sequencing Platform Introduce Bias in Metagenomic-Based Characterizations of Microbiomes
Casper S. Poulsen, Claus T. Ekstrøm, Frank M. Aarestrup, Sünje J. Pamp
bioRxiv 592154; doi: https://doi.org/10.1101/592154

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