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Sci Rep. 2018 Sep 6;8(1):13377. doi: 10.1038/s41598-018-31420-7.

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses.

Author information

1
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
2
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
3
University of Wisconsin Oshkosh, Oshkosh, WI, 54901, USA.
4
Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, 90502, USA.
5
New England Biolabs, Ipswich, MA, 01938, USA.
6
Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
7
David Geffen School of Medicine at UCLA, Los Angeles, California, 90502, USA.
8
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA. jdhotopp@som.umaryland.edu.
9
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA. jdhotopp@som.umaryland.edu.
10
Greenebaum Cancer Center, University of Maryland, Baltimore, MD, 21201, USA. jdhotopp@som.umaryland.edu.

Abstract

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r2 = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2 = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies.

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