Target-Enriched Endosymbiont Sequencing (TEEseq): A New High-Throughput Sequencing Approach Applied to the Comprehensive Characterization of Endosymbionts

  • Hannes SchulerEmail author
  • Jacqueline A. Lopez
  • Meredith M. Doellman
  • Glen R. Hood
  • Scott P. Egan
  • Jeffrey L. Feder
Part of the Methods in Molecular Biology book series (MIMB, volume 1858)


Intracellular bacteria are ubiquitous in the insect world, with perhaps the best-studied example being the alphaproteobacterium, Wolbachia. Like most endosymbionts, Wolbachia cannot be cultivated outside of its host cells, hindering traditional microbial characterization techniques. Furthermore, multiple Wolbachia strains can be present within a single host, and certain strains can be present in densities below the detection limit of current methods. To date, Wolbachia has most commonly been studied using polymerase chain reaction (PCR) amplification and Sanger DNA sequencing by targeting specific genes in the bacterium’s genome. PCR amplification and Sanger sequencing of multiple Wolbachia strains requires analysis of individually cloned sequences, which is resource and labor intensive. To help mitigate these difficulties, we present a modified double digest restriction site associated DNA sequencing (ddRADseq) approach to target and sequence in parallel multiple genes by adding restriction enzyme recognition sites to gene-specific PCR primers. Adopting this strategy allows us to uniquely tag and sequence amplicons from multiple hosts simultaneously on an Illumina MiSeq platform. Our approach represents an efficient and cost-effective method to characterize multiple target genes in population surveys.

Key words

Wolbachia Endosymbiont Microbial community Metabarcoding PCR enrichment ddRAD Illumina sequencing 



We thank Melissa Stephens, Michael Pfrender, and Stuart Jones for helpful discussions. HS is supported by the Erwin Schrödinger Fellowship J-3527-B22 of the Austrian Science Fund FWF. In addition, the work was aided by support to JLF from the National Science Foundation and United States Department of Agriculture.

Supplementary material

436248_1_En_14_MOESM1_ESM.xlsx (54 kb)
Supplemental Table 1 (XLSX 53 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Hannes Schuler
    • 1
    • 2
    Email author
  • Jacqueline A. Lopez
    • 3
  • Meredith M. Doellman
    • 1
  • Glen R. Hood
    • 1
    • 4
  • Scott P. Egan
    • 5
  • Jeffrey L. Feder
    • 1
  1. 1.Department of Biological Sciences, Galvin Life Sciences BuildingUniversity of Notre DameNotre DameUSA
  2. 2.Faculty of Science and TechnologyFree University of Bozen-BolzanoBolzanoItaly
  3. 3.Genomics and Bioinformatics Core FacilityUniversity of Notre DameNotre DameUSA
  4. 4.Department of Biological SciencesWayne State UniversityDetroitUSA
  5. 5.Department of BioSciencesRice UniversityHoustonUSA

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