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Enrichment strategy for sequencing of Wolbachia genomes from isopod crustaceans
Myriam Badawi, Isabelle Giraud, Pierre Greve, Richard Cordaux
To cite this version:
Myriam Badawi, Isabelle Giraud, Pierre Greve, Richard Cordaux. Enrichment strategy for sequencing of Wolbachia genomes from isopod crustaceans. 7th International Wolbachia Conference, Jun 2012, Oléron, France. 2012. �hal-01622152�
Enrichment strategy for sequencing of Wolbachia genomes from
isopod crustaceans
Enrichment strategy for sequencing of Wolbachia genomes from
isopod crustaceans
Myriam BADAWI, Isabelle GIRAUD, Pierre GREVE, Richard CORDAUX
Université de Poitiers, Laboratoire Ecologie et Biologie des Interactions - UMR CNRS 7267, Equipe Ecologie Evolution Symbiose - Bat. B8, 40 avenue du Recteur Pineau F-86022 POITIERS Cedex FRANCE myriam.badawi@univ-poitiers.fr
Enrichment method:
Conclusion: This method of selective enrichment of Wolbachia DNA is efficient, reproducible and provides sufficient amounts of Wolbachia DNA for high-throughput sequencing. Because it avoids centrifugations, DNaseI treatment and pulse- field electrophoresis, it may easily be extended to other Wolbachia strains of isopod crustaceans.
Dissection of ovaries
Shred cells but not nuclei with a dounce in PBS solution
Filtration 5µm
454 Sequencing
Without enrichment With enrichment
Proportion of wCon nucleotides in 454 sequencing data
Centrifugations
DNaseI treatment
Pulse-field electrophoresis
Sanger sequencing
Feminization
Cytoplasmic incompatibility
Closely related
Context: Wolbachia alters the reproduction of many arthropods. Notably, in isopod crustacean species, it can induce feminization or cytoplasmic incompatibility (Cordaux et al., 2004 ; Moret et al., 2001). Our ultimate goal is to determine the gene(s) involved in feminization. To reach this goal, we chose to use a comparative genomics strategy between a feminizing strain (wVulC) and a closely related one (wCon) that induces cytoplasmic incompatibility on its host.
Results: Enrichment estimation by qPCR for wCon
Three genomes typically present in an isopod cell infected by Wolbachia
Nucleus
Mitochondria Wolbachia
Nucleus
Nucleus
wCon
Mitochondria Mitochondria
wCon
Relative quantities of nuclear, mitochondrial and wCon DNA.
Average proportions among 4 different pools (5-30 individuals) without enrichment and 8 different pools with enrichment.
Challenge:
in a classical total DNA extraction, Wolbachia DNA is contaminated by
nuclear and mitochondrial DNA Challenge:
in a classical total DNA extraction, Wolbachia DNA is contaminated by
nuclear and mitochondrial DNA
Unknown genetic basis of feminization
Unknown genetic basis of feminization
Comparative genomics Comparative
genomics
wVulC
(host: Armadillidium vulgare) wVulC
(host: Armadillidium vulgare)
(host: Cylisticus convexus)wCon wCon
(host: Cylisticus convexus)
GENOME SEQUENCED GENOME SEQUENCED
Objective: To purify DNA of wCon with a technique which :
● reproducibly provides enough DNA quantity for sequencing,
● is easy to adapt to other terrestrial isopod species.
→ We would like to avoid time-consuming steps which require optimization when changing the host species.
→ As a reference genome is available (wVulC), we don't necessarily need highly enriched Wolbachia DNA.
Method previously used to purify wVulC
Method previously
used to purify wVulC Method we used to purify wCon Method we used
to purify wCon
→ wCon DNA enrichment: ~300x
→ Reproducible
→ wCon DNA enrichment: ~300x
→ Reproducible
GENOME
TO BE SEQUENCED GENOME
TO BE SEQUENCED
Nucleus
wCon
Mitochondria
Relative quantities of nuclear, mitochondrial and wCon DNA of
the sequenced sample
→ Consistent proportion of wCon DNA with qPCR estimates
→ Data usable for comparative genomics
→ Consistent proportion of wCon DNA with qPCR estimates
→ Data usable for comparative genomics
● ~60Mb of DNA sequences obtained with ¼ 454 run
● Reads mapped against Wolbachia genomes and A. vulgare mitochondrial genome, using gsMapper
Remove nuclear DNA
Remove nuclear DNA and mitochondrial DNA
Remove
mitochondrial DNA
Highly enriched wVulC DNA Highly enriched
wVulC DNA Moderately enriched wCon DNA
Moderately enriched wCon DNA
End product
References :
Cordaux R et al., (2004) Evidence for a new feminizing Wolbachia strain in the isopod Armadillidium vulgare: evolutionary implications. Heredity 93: 78-84.
Moret Y et al., (2001) Wolbachia endosymbiont responsible for cytoplasmic incompatibility in a terrestrial crustacean: effects in natural and foreign hosts. Heredity 86: 325-332.
Contact :
myriam.badawi@univ-poitiers.fr
© tom murray
© Petr Pivoňka
