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Title: Evaluation of the Effects of Library Preparation Procedure and Sample Characteristics on the Accuracy of Metagenomic Profiles
ABSTRACT Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera Flex kit, perform when applied to metagenomic DNA. We measured each method’s ability to produce metagenomic data that accurately represent the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral associated, and mouse gut associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each method’s performance, indicating that careful consideration may be needed when selecting between methods, especially for low-complexity communities. However, the cost-effective preparation methods that we assessed are generally comparable to the current gold-standard Nextera DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies. IMPORTANCE Metagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely accepted standardized library preparation methods can be cost-prohibitive. Newly available approaches may be less expensive, but their efficacy in comparison to standardized methods remains unknown. In this study, we compared five different metagenomic library preparation methods. We evaluated each method across a range of microbial communities varying in complexity and quantity of input DNA. Our findings demonstrate the importance of considering sample properties, including community type, composition, and DNA amount, when choosing the most appropriate metagenomic library preparation method.  more » « less
Award ID(s):
2025457
PAR ID:
10310950
Author(s) / Creator(s):
; ; ; ; ;
Editor(s):
David, Lawrence A.
Date Published:
Journal Name:
mSystems
Volume:
6
Issue:
5
ISSN:
2379-5077
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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    Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).

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    Results

    Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from theMicroviridaefamily, can be among the most abundant viral genomes in a sample.

    Discussion

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Retrieved from https://par.nsf.gov/biblio/10310950. <em>mSystems</em> 6.5 Web. doi:10.1128/mSystems.00440-21. </div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="links-format"><a href="#cite-apa" data-toggle="modal">APA</a> <div id="cite-apa" class="modal" tabindex="-1" role="dialog" aria-labelledby="cite-apa_label" aria-hidden="true"> <div class="modal-dialog"> <div class="modal-content"> <div class="modal-header"> <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button> <strong id="cite-apa_label">Cite: APA Format</strong> </div> <div class="modal-body">Gaulke, Christopher A., Schmeltzer, Emily R., Dasenko, Mark, Tyler, Brett M., Vega Thurber, Rebecca, & Sharpton, Thomas J. <em>Evaluation of the Effects of Library Preparation Procedure and Sample Characteristics on the Accuracy of Metagenomic Profiles</em>. <em>mSystems</em>, <em>6</em> (5). Retrieved from https://par.nsf.gov/biblio/10310950. <a href="https://doi.org/10.1128/mSystems.00440-21">https://doi.org/10.1128/mSystems.00440-21</a> </div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="links-format"><a href="#cite-chi" data-toggle="modal">Chicago</a> <div id="cite-chi" class="modal" tabindex="-1" role="dialog" aria-labelledby="cite-chi_label" aria-hidden="true"> <div class="modal-dialog"> <div class="modal-content"> <div class="modal-header"> <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button> <strong id="cite-chi_label">Cite: Chicago Format</strong> </div> <div class="modal-body">Gaulke, Christopher A., Schmeltzer, Emily R., Dasenko, Mark, Tyler, Brett M., Vega Thurber, Rebecca, and Sharpton, Thomas J. "Evaluation of the Effects of Library Preparation Procedure and Sample Characteristics on the Accuracy of Metagenomic Profiles". <em>mSystems</em> 6 (5). Country unknown/Code not available. <a href="https://doi.org/10.1128/mSystems.00440-21">https://doi.org/10.1128/mSystems.00440-21.</a> <a href="https://par.nsf.gov/biblio/10310950">https://par.nsf.gov/biblio/10310950</a>. </div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="links-format"><a href="#cite-bib" data-toggle="modal">BibTeX</a> <div id="cite-bib" class="modal" tabindex="-1" role="dialog" aria-labelledby="cite-bib_label" aria-hidden="true"> <div class="modal-dialog"> <div class="modal-content"> <div class="modal-header"> <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button> <strong id="cite-bib_label">Cite: BibTeX Format</strong> </div> <div class="modal-body"> @article{osti_10310950,<br/> place = {Country unknown/Code not available}, title = {Evaluation of the Effects of Library Preparation Procedure and Sample Characteristics on the Accuracy of Metagenomic Profiles}, url = {https://par.nsf.gov/biblio/10310950}, DOI = {10.1128/mSystems.00440-21}, abstractNote = {ABSTRACT Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera Flex kit, perform when applied to metagenomic DNA. We measured each method’s ability to produce metagenomic data that accurately represent the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral associated, and mouse gut associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each method’s performance, indicating that careful consideration may be needed when selecting between methods, especially for low-complexity communities. However, the cost-effective preparation methods that we assessed are generally comparable to the current gold-standard Nextera DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies. IMPORTANCE Metagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely accepted standardized library preparation methods can be cost-prohibitive. Newly available approaches may be less expensive, but their efficacy in comparison to standardized methods remains unknown. In this study, we compared five different metagenomic library preparation methods. We evaluated each method across a range of microbial communities varying in complexity and quantity of input DNA. Our findings demonstrate the importance of considering sample properties, including community type, composition, and DNA amount, when choosing the most appropriate metagenomic library preparation method.}, journal = {mSystems}, volume = {6}, number = {5}, author = {Gaulke, Christopher A. and Schmeltzer, Emily R. and Dasenko, Mark and Tyler, Brett M. and Vega Thurber, Rebecca and Sharpton, Thomas J.}, editor = {David, Lawrence A.} }</div> <div class="modal-footer"> <button class="btn btn-sm btn-default" data-dismiss="modal" aria-hidden="true">Close</button> </div> </div> </div> </div></li> <li class="divider"></li> </ul> <ul class="nav nav-list" style="font-size: 14px; font-family: Arial Regular;"> <li class="nav-header header-format">Export Metadata</li> <li class="links-format"><a href="https://par.nsf.gov/endnote?osti_id=10310950">EndNote</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:excel/osti-id:10310950">Excel</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:csv/osti-id:10310950">CSV</a></li> <li class="links-format"><a href="https://par.nsf.gov/export/format:xml/osti-id:10310950">XML</a></li> <li class="divider"></li> </ul> <ul class="nav nav-list" style="font-size: 14px; 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