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Member Symposium

Systematics, Evolution, and Biodiversity

Plant-Insect Ecosystems

Down a Long and Winding Road: Tackling Insect Research Using Long Reads

Direct sequencing of insect symbionts via nanopore adaptive sampling

Tuesday, November 12, 2024
8:05 AM – 8:23 AM MST
Location: Phoenix Convention Center, 226 A
Oral Presentation
Insect symbionts can significantly influence host phenotype, ranging from beneficial to pathogenic effects, with many insects harboring multiple co-infections, complicating their study. However, less than 1% of insect species have high-quality referenced genomes available, and even fewer have their symbionts sequenced. In many cases research into these symbionts is limited as they cannot be cultured, are not present in a high enough population to be identified in lower-coverage sequencing studies and/or, are not well characterized in the literature or databases. Adaptive sampling, a feature of ONT, enables real-time selection or rejection of long-read sequences during sequencing runs. Leveraging this capability, we developed a subtractive adaptive sampling method to sequence the complete genomes of mitochondria, Buchnera, and its plasmids (pLeu, pTrp), as well as Wolbachia genomes in two aphid species, Aphis glycines and Pentalonia nigronervosa. gDNA was extracted from ~10 aphids and a fastq file representing the host (either Aphis glycines or Pentalonia nigronervosa) was provided to the MinKnow software. Reads that mapped to the host genome were rejected from the MinION flowcell in real-time, while other reads were sequenced. This approach resulted in more than 100-fold enrichment of the genomes of interest over the background, and each genome was assembled into one complete contig. In the case of the plasmids, individual nanopore reads spanned the entire molecule. These reads revealed the exact number of operons present on each plasmid, a detail not possible via short-read methods.