Unamplified, Long-Read Metagenomic Sequencing Approach to Close Endosymbiont Genomes of Low-Biomass Insect Populations
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 system...
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| Format: | Article |
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MDPI AG
2022-02-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/10/3/513 |
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| author | Joseph R. Petrone Alam Muñoz-Beristain Paula Rios Glusberger Jordan T. Russell Eric W. Triplett |
| author_facet | Joseph R. Petrone Alam Muñoz-Beristain Paula Rios Glusberger Jordan T. Russell Eric W. Triplett |
| author_sort | Joseph R. Petrone |
| collection | DOAJ |
| description | 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 <i>Diaphorina citri</i> 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 <i>D. citri</i> 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 <i>Wolbachia</i> endosymbiont of <i>Diaphorina citri</i> 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. |
| first_indexed | 2024-03-09T13:16:13Z |
| format | Article |
| id | doaj.art-32d52bb3d99f41f3b0899246a6f6fcd1 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-09T13:16:13Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-32d52bb3d99f41f3b0899246a6f6fcd12023-11-30T21:35:19ZengMDPI AGMicroorganisms2076-26072022-02-0110351310.3390/microorganisms10030513Unamplified, Long-Read Metagenomic Sequencing Approach to Close Endosymbiont Genomes of Low-Biomass Insect PopulationsJoseph R. Petrone0Alam Muñoz-Beristain1Paula Rios Glusberger2Jordan T. Russell3Eric W. Triplett4Microbiology and Cell Science Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32603, USAMicrobiology and Cell Science Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32603, USAMicrobiology and Cell Science Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32603, USAMicrobiology and Cell Science Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32603, USAMicrobiology and Cell Science Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32603, USAWith 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 <i>Diaphorina citri</i> 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 <i>D. citri</i> 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 <i>Wolbachia</i> endosymbiont of <i>Diaphorina citri</i> 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.https://www.mdpi.com/2076-2607/10/3/513psyllidinsect metagenomenext-generation sequencingOxford NanoporePacBiolow-biomass |
| spellingShingle | Joseph R. Petrone Alam Muñoz-Beristain Paula Rios Glusberger Jordan T. Russell Eric W. Triplett Unamplified, Long-Read Metagenomic Sequencing Approach to Close Endosymbiont Genomes of Low-Biomass Insect Populations Microorganisms psyllid insect metagenome next-generation sequencing Oxford Nanopore PacBio low-biomass |
| title | Unamplified, Long-Read Metagenomic Sequencing Approach to Close Endosymbiont Genomes of Low-Biomass Insect Populations |
| title_full | Unamplified, Long-Read Metagenomic Sequencing Approach to Close Endosymbiont Genomes of Low-Biomass Insect Populations |
| title_fullStr | Unamplified, Long-Read Metagenomic Sequencing Approach to Close Endosymbiont Genomes of Low-Biomass Insect Populations |
| title_full_unstemmed | Unamplified, Long-Read Metagenomic Sequencing Approach to Close Endosymbiont Genomes of Low-Biomass Insect Populations |
| title_short | Unamplified, Long-Read Metagenomic Sequencing Approach to Close Endosymbiont Genomes of Low-Biomass Insect Populations |
| title_sort | unamplified long read metagenomic sequencing approach to close endosymbiont genomes of low biomass insect populations |
| topic | psyllid insect metagenome next-generation sequencing Oxford Nanopore PacBio low-biomass |
| url | https://www.mdpi.com/2076-2607/10/3/513 |
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