Abstract
With the current advancements in DNA sequencing technology, the limiting factor in long-read metagenomic assemblies is now the quantity and quality of input DNA. Although these requirements can be met through the use of axenic bacterial cultures or large amounts of biological material, insect systems that contain unculturable bacteria or that contain a low amount of available DNA cannot fully utilize the benefits of third-generation sequencing. The citrus greening disease insect vector Diaphorina citri is an example that exhibits both of these limitations. Although endosymbiont genomes have mostly been closed after the short-read sequencing of amplified template DNA, creating de novo long-read genomes from the unamplified DNA of an insect population may benefit communities using bioinformatics to study insect pathosystems. Here all four genomes of the infected D. citri microbiome were sequenced to closure using unamplified template DNA and two long-read sequencing technologies. Avoiding amplification bias and using long reads to assemble the bacterial genomes allowed for the circularization of the Wolbachia endosymbiont of Diaphorina citri for the first time and paralleled the annotation context of all four reference genomes without utilizing a traditional hybrid assembly. The strategies detailed here are suitable for the sequencing of other insect systems for which the input DNA, time, and cost are an issue.
Highlights
Studying the endosymbiont populations of non-model insects can be challenging due to the lack of ideal conditions and available information on organism-specific extraction optimizations
To generate enough endosymbiont gDNA for long-read sequencing to capture the four genomes, insects first needed to be selectively pooled from individual insect extractions of those that were infected with the highest titer of the limited bacterium, Ca
As for the other long-read technology, the prepared Pacific Biosciences (PacBio) library was run on a single SMRT cell lane for 1200 min, resulting in 34.8 Gb and 1.33 × 107 subreads, with a median subread length of 3.11 Kb
Summary
Studying the endosymbiont populations of non-model insects can be challenging due to the lack of ideal conditions and available information on organism-specific extraction optimizations. These setbacks can appear due to a lack of sample availability, DNA extraction yield and quality, or an irregularly dispersed bacterial species of interest among an infected insect population [1]. In cases where the DNA yield from a single insect is below the requirement for next-generation sequencing (NGS), such as mosquitos, whiteflies, aphids, ticks, and psyllids [1,3–9], whole genome amplification (WGA) may be required to obtain enough. This amplification process can introduce bias through inherent polymerase error rates, exclusion biases of smaller and low-abundance fragments [11], and the amplification of contamination in metagenomic sequencing [12]. PCRbased WGA methods can lead to uneven genome coverage and incorrect assemblies [13], and these risks increase with AT-rich genomes [14]
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