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Mining Eukaryotic Meta-Genomes for Endosymbionts using Next-Generation Sequencing
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Mining Eukaryotic Meta-Genomes for Endosymbionts using Next-Generation Sequencing


The availability of high-throughput next generation sequencing technologies presents an opportunity for in-silico discovery of endosymbionts. We describe a method for mining a whole genome shotgun …

The availability of high-throughput next generation sequencing technologies presents an opportunity for in-silico discovery of endosymbionts. We describe a method for mining a whole genome shotgun metagenome to identify members of the endosymbiont community followed by reconstruction and validation of a high-quality draft microbial genome.
The Asian citrus psyllid (D. citri Kuwayama or ACP) is host to 7+ bacterial endosymbionts and is the insect vector of Ca. liberibacter asiaticus, causal agent of citrus greening, a disease that has cost the Florida citrus industry $3.63 billion since 2006.
DNA from D. citri was sequenced to 108X coverage to produce paired-end and mate-pair Illumina libraries. The sequences were mined for wolbachia (wACP) reads using 4 sequenced Wolbachia genomes as bait. Putative wACP reads were then assembled using Velvet and MIRA3 assemblers. The resulting wACP contigs were annotated using the RAST and compared to the closest sequenced wolbachia from an insect genome, Wolbachia endosymbiont of Culex quinquefasciatus (wPip). MIRA3 was able to reconstruct a majority of the wPip CDS regions and was therefore, selected for scaffolding using large insert mate-pair libraries. The wACP scaffolds were further improved using wPip as reference genome to orient and order the contigs.
In order to determine the presence of the core Wolbachia proteins in our wACP scaffold, we compared them to core Wolbachia proteins identified by OrthoMCL. 1164/1213 wACP proteins had matches of which 669 were to core proteins. This number compares favorably to the number of core proteins (670) found in sequenced Wolbachias.

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  • 1. Mining Eukaryotic Meta-Genomesfor EndosymbiontsExploring the Asian citrus psyllid microbiomeSurya Saha1, Wayne B. Hunter2 andMagdalen Lindeberg 11 Department of Plant Pathology and Plant-Microbe Biology, CornellUniversity, Ithaca, NY, USA, 2 USDA-ARS, US Horticultural Research Lab, FortPeirce, FL, USAGLBio 2013, Pittsburgh
  • 2. You are free to:Copy, share, adapt, or re-mix;Photograph, film, or broadcast;Blog, live-blog, or post video of;This presentation. Provided that:You attribute the work to its author andrespect the rights and licensesassociated with its components.Slide Concept by Cameron Neylon: : @SahaSuryaGLBio 2013, Pittsburgh
  • 3. AcknowledgementsFundingMagdalen LindebergCornell University• J. Kent Morgan, USDA-ARS• Mizuri Marutani-Hert, USDA-ARS• Hong Huang, University of South FloridaWayne HunterUSDA-ARSJustin ReeseGenformatic, LLC.GLBio 2013, Pittsburgh
  • 4. Figure 1. Workflow used to identify regions of reference genomes illuminated bysequence reads from the Diaphorina citri metagenome data set.
  • 5. HLB found in Florida2005Worldwide distribution of CitrusGreening/Huanglongbing1st scientificreport of HLB inChina – 1919GLBio 2013, Pittsburgh
  • 6. What is the impact of this disease?Citrus – a 9.3 billion dollar industry in Florida and 1.2 billion dollars in California• Leave agricultural practices unchanged - significant loss of production• Begin expensive , labor intensive controls - increase cost of productionhealthy infectedIncreased cost of citrus products for consumersBetween 2006 and 2011 greening had cost Florida$4.5 billion in lost economic output, and 8,257 jobs.University of Florida report, 2013 2013, Pittsburgh
  • 7. How it gets around:Ca. Liberibacter asiaticus is vectored byAsian Citrus PsyllidInsect transmission greatly enhancespotential for rapid long distance spreadPsyllid feeds on many members of the citrus familyOnce infected, each plant becomes a disease reservoir fromwhich uninfected psyllids can pick up bacteriaCa. Liberibacter asiaticusin citrus phloem cellsGLBio 2013, Pittsburgh
  • 8. Liberibacterpsyllid citrusBig picture for molecular interactionsCould lead to identification ofmetabolically complementarybacteria for co-culturing withLiberibacterUnculturableGLBio 2013, Pittsburgh
  • 9. Why endosymbionts? Significant impact on diverse hostprocesses Nutritional status Reproduction Lifespan Resistance to insecticides Means to explore population dynamicsand relatedness to other isolates Known examples of highly-evolvedhost-symbiont relationship Buchnera and Aphids Psyllid microbiome diversity 10 bacteria reported in ACP (Florida) 5 additional bacteria reported inclosely related potato psyllid (B.cockerelli)Buchnera-derived essential aminoacids supporting the growth of peaaphid larvae. [ Gündüz and Douglas2009]GLBio 2013, Pittsburgh
  • 10. Candidate endosymbiontsDescription Length (bp) CoverageLength(bp)Klebsiella variicola At-22 chromosome, completegenome 5,458,505 1.144% 307,111Salmonella enterica subsp. enterica serovar Typhi str.Ty2 chromosome, complete genome 4,791,961 1.266% 464,747Staphylococcus epidermidis ATCC 12228chromosome, complete genome 2,499,279 3.077% 60,415Acidovorax avenae subsp. avenae ATCC 19860chromosome, complete genome 5,482,170 0.473% 71,218Acinetobacter sp. DR1 chromosome,completegenome 4,152,543 2.763% 102,446Herbaspirillum seropedicae SmR1 chromosome,complete genome 5,513,887 0.949% 23,509Wolbachia endosymbiont of Culex quinquefasciatusPel, complete genome 1,482,455 86.787% 1,140,899Methylibium petroleiphilum PM1 chromosome,complete genome 4,044,195 0.318% 20,107Why Wolbachia? Known obligate association with up to 16% of insectsspecies Positive correlation of titer with Ca. liberibacer asiaticus Associated with Cytoplasmic Incompatibility in hosts Used as biocontrol in dengue fever and malariaGLBio 2013, Pittsburgh
  • 11. Read capture using baits Wolbachia endosymbiont of Drosophila melanogaster Wolbachia endosymbiont of Culex quinquefasciatus Pel (wPip) Wolbachia sp. wRi Wolbachia endosymbiont strain TRS of Brugia malayiDataset Total (bp) 100% 90% 85%Paired-end 33,708,218,600 127,335,000 228,893,000 237,906,000Mate-pair 2k 7,020,642,800 11,200 45,200 63,000Mate-pair 5k 6,000,789,600 10,000 43,000 66,800Mate-pair 10k 7,307,423,600 2,895,000 4,427,600 4,909,000Results of mining putative wDi reads from the ACP metagenomeGLBio 2013, Pittsburgh
  • 12. Assembler Contigs N50Contigs >1kLength ofContigs >40kVelvetOptimizer(n50, 83 mer)111 79935 53 807171VelvetOptimizer(n50/tbp, 95 mer)118 84957 25 744357MIRA3 167 23068 105 247976Annotation0 1000 2000Total CDSRNAFunctional - shared with wPipFunctional - wPip aloneFunctional - not in wPipwPipMIRA3VelvetOptimizer (n50/tbp, 95 mer)VelvetOptimizer (n50, 83 mer)ScaffoldingDescription Contigs Longest N50Contigs >1kSOPRA / wDi2 101 89608 41594 77Minimus2 /wDi199 106898 41594 76Minimus2 /wDi3104 206691 52418 66WorkflowwDi1 wDi2 wDi3Total CDS 1176 1190 1183RNA 36 36 36Functional - sharedwith wPip608 610 608Functional - wPipalone24 23 24Functional - not inwPip23 22 23Functionalannotationusing RASTGLBio 2013, Pittsburgh
  • 13.  660 core protein clusters per Wolbachia genome 659 core protein cluster representatives found in wDi Missing core cluster consists entirely of hypotheticals 32 lineage specific genes in wDi w.r.t. wPip All have unknown function 11 have homologs in other endosymbionts of mosquitoWolbachia endosymbiont of Drosophila melanogasterWolbachia wPip endosymbiont of Culex quinquefasciatus PelWolbachia sp. wRiWolbachia endosymbiont strain TRS of Brugia malayi0200400600800100012001400Total proteinsCoreCoreparalogousSharedSharedparalogous Lineagespecific LineagespecificparalogousOrthoMCL analysis of pan-proteome Core / Core paralogous Shared / Shared paralogous Lineage specificGLBio 2013, Pittsburgh
  • 14. Conclusions Targeted genome reconstruction strategy for endosymbionts Recommendations MIRA3 produced more fragmented but qualitatively better assembliesthan Velvet SOPRA and SSPACE are effective for scaffolding when coupled withMinimus2 Mapped regions on reference should be validated for uniqueness Screening out ribosomal regions prior to mapping reduces falsepositives Caveats Missing lineage specific regions Low throughput methodGLBio 2013, Pittsburgh
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  • 16. Questions??Slides :