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Accurate Microbiome Sequencing with Synthetic Long Read Sequencing

View ORCID ProfileNico Chung, View ORCID ProfileMarc W. Van Goethem, View ORCID ProfileMelanie A. Preston, Filip Lhota, Leona Cerna, View ORCID ProfileFerran Garcia-Pichel, Vanessa Fernandes, View ORCID ProfileAna Giraldo-Silva, Hee Shin Kim, Evan Hurowitz, Michael Balamotis, Indira Wu, Tuval Ben-Yehezkel
doi: https://doi.org/10.1101/2020.10.02.324038
Nico Chung
1Welgene Biotech, Taipei, Taiwan
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Marc W. Van Goethem
2Lawrence Berkeley National Laboratory, Berkeley, CA, USA
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Melanie A. Preston
3Promega Corporation, Madison, WI, USA
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Filip Lhota
4Gennet, Centre for Medical Genetics and Reproductive Medicine, Prague, Czech Republic
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Leona Cerna
4Gennet, Centre for Medical Genetics and Reproductive Medicine, Prague, Czech Republic
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Ferran Garcia-Pichel
5Center for Fundamental and Applied Microbiomics, Arizona State University, AZ, USA
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Vanessa Fernandes
5Center for Fundamental and Applied Microbiomics, Arizona State University, AZ, USA
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Ana Giraldo-Silva
5Center for Fundamental and Applied Microbiomics, Arizona State University, AZ, USA
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  • ORCID record for Ana Giraldo-Silva
Hee Shin Kim
6Loop Genomics, San Jose, CA, USA
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Evan Hurowitz
6Loop Genomics, San Jose, CA, USA
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Michael Balamotis
6Loop Genomics, San Jose, CA, USA
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Indira Wu
6Loop Genomics, San Jose, CA, USA
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Tuval Ben-Yehezkel
6Loop Genomics, San Jose, CA, USA
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  • For correspondence: tuval@loopgenomics.com
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Abstract

The microbiome plays a central role in biochemical cycling and nutrient turnover of most ecosystems. Because it can comprise myriad microbial prokaryotes, eukaryotes and viruses, microbiome characterization requires high-throughput sequencing to attain an accurate identification and quantification of such co-existing microbial populations. Short-read next-generation-sequencing (srNGS) revolutionized the study of microbiomes and remains the most widely used approach, yet read lengths spanning only a few of the nine hypervariable regions of the 16S rRNA gene limit phylogenetic resolution leading to misclassification or failure to classify in a high percentage of cases. Here we evaluate a synthetic long-read (SLR) NGS approach for full-length 16S rRNA gene sequencing that is high-throughput, highly accurate and low-cost. The sequencing approach is amenable to highly multiplexed sequencing and provides microbiome sequence data that surpasses existing short and long-read modalities in terms of accuracy and phylogenetic resolution. We validated this commercially-available technology, termed LoopSeq, by characterizing the microbial composition of well-established mock microbiome communities and diverse real-world samples. SLR sequencing revealed differences in aquatic community complexity associated with environmental gradients, resolved species-level community composition of uterine lavage from subjects with histories of misconception and accurately detected strain differences, multiple copies of the 16S rRNA in a single strain’s genome, as well as low-level contamination in soil cyanobacterial cultures. This approach has implications for widespread adoption of high-resolution, accurate long-read microbiome sequencing as it is generated on popular short read sequencing platforms without the need for additional infrastructure.

Competing Interest Statement

Hee Shin Kim, Evan Hurowitz, Michael Balamotis, Indira Wu, Tuval Ben-Yehezkel are employees of Loop Genomics, the vendor for the synthetic long-read sequencing technology analyzed in this manuscript.

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 October 03, 2020.
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Accurate Microbiome Sequencing with Synthetic Long Read Sequencing
Nico Chung, Marc W. Van Goethem, Melanie A. Preston, Filip Lhota, Leona Cerna, Ferran Garcia-Pichel, Vanessa Fernandes, Ana Giraldo-Silva, Hee Shin Kim, Evan Hurowitz, Michael Balamotis, Indira Wu, Tuval Ben-Yehezkel
bioRxiv 2020.10.02.324038; doi: https://doi.org/10.1101/2020.10.02.324038
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Accurate Microbiome Sequencing with Synthetic Long Read Sequencing
Nico Chung, Marc W. Van Goethem, Melanie A. Preston, Filip Lhota, Leona Cerna, Ferran Garcia-Pichel, Vanessa Fernandes, Ana Giraldo-Silva, Hee Shin Kim, Evan Hurowitz, Michael Balamotis, Indira Wu, Tuval Ben-Yehezkel
bioRxiv 2020.10.02.324038; doi: https://doi.org/10.1101/2020.10.02.324038

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