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Poster # P66 and Their Wolbachia Endosymbionts Amit Sinha1*, Catherine B

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Poster # P66 and Their Wolbachia Endosymbionts Amit Sinha1*, Catherine B Draft genome sequences of Mansonella perstans and Mansonella ozzardi Poster # P66 and their Wolbachia endosymbionts Amit Sinha1*, Catherine B. Poole1*, Rick D. Morgan1, Zhiru Li1, Laurence Ettwiller1, Marcelo U. Ferreira2, Nathália F. Lima2, Samuel Wanji3, ClotilDe K.S. Carlow1 1New EnglanD Biolabs, Ipswich, MA, USA. 2 University of São Paolo, Brazil. 3 University of Cameroon, Buea, Cameroon. * = Equal contribution Mansonelliasis : A neglected filarial infection Genome Assembly Pipeline Genome comparisons • The two Mansonella genomes are largely colinear • Orthology analysis • Mansonelliasis is a widespread yet neglected Remove reads that map Raw sequencing reads QC and Pre-processing filariasis of humans, caused by infection with any of (A) to the human genome • Genome-wide Average Nucleotide Identity, gANI ~ 90%. identified the set of shared the three filarial species: Mansonella perstans, Dot-plot of whole genome alignments using MUMmer and unique filarial genes. Unique forward alignments M. ozzardi and M. streptocerca. Reads that did not Unique reverse alignments Assemble & Mansonella Repetitive alignments map to any B. malayi 3,473 • Annotate genome reads n = 11,019 M. perstans infections are endemic in Central and Wolbachia genome 400 Potential Mansonella 263 West Africa (Figure 1A), and in a few areas South scaffolds & Wolbachia reads 6,883 America (stippled region in Figure 1B). The migration Reads that map to Assemble & Wolbachia 2,671 1,346 M. perstans any Wolbachia M. ozzardi 490 of M. perstans from Africa to South America is most Annotate genome reads M. perstans M. ozzardi genome n = 10,307 n = 9,119 likely a consequence of slave trade. Modified from: M. perstans scaffolds • M. ozzardi infections are highly prevalent in South Simonsen et al. 2011 America and the Caribbean islands (Figure 1B). (B) Mansonella Genome : Draft Assemblies Completeness of Wolbachia Genomes from Mansonella • Transmission to humans is via insect vectors such as Wolbachia Wolbachia Dot-plot of whole genome alignments using MUMmer M. perstans M. ozzardi • Both wMpe and wMoz genomes show the biting midges of Culicoides spp. for M. perstans wMpe wMoz Unique forward alignments high sequence identity (gANI ~ 93%), and Unique reverse alignments Repetitive alignments and M. ozzardi, and additionally via black flies of Venezuela Sample Name Mp2 Mo2 Mp1 Mo2 Columbia collinearity with the bed-bug Wolbachia Simulium spp. for M. ozzardi. Sequencing Platform PacBio Illumina Illumina Illumina wCle. scaffolds wMoz • The Wolbachia endosymbionts of other filaria are M. perstans § Estimated genome size 75 to 85 Mb * 75 to 85 Mb * 0.8 to 1.2 Mb 0.8 to 1.2 Mb § placed in supergroups C, D and J in Wolbachia Assembled genome size 79 Mb 80 Mb 0.70 Mb 1.5 Mb • The Wolbachia wMoz genome is nearly phylogeny (Lefoulon et al. 2016). Mansonella spp. wCle genome M. ozzardi Number of scaffolds 1,004 4,912 550 523 complete, with BUSCO score of ~80%, filariae are unique as their endosymbiont Wolbachia Modified from: similar to other completely sequenced wMoz is a member of supergroup F, the only clade Lima et al. 2016 Largest scaffold 1.24 Mb 550 kb 5.5 kb 48 kb Wolbachia genomes. wMpe containing Wolbachia from both insect and filarial Figure 1: Geographic ranges of (A) M. Scaffold N50 size 227 kb 73 kb 1.4 kb 10 kb perstans and (B) M. ozzardi. The stippled wCle hosts. %GC 30% 30% 36 % 34 % region in (B) represents areas where both • wOv M. ozzardi and M. perstans have been However, the Wolbachia wMpe genome • The goal of the current study was to obtain genome Predicted genes 10,307 9,119 885 1,871 wBm reported to occur. The red pins mark the shows a large fraction of fragmented and sequence and assemblies of M. perstans and origin of samples used in this study. wMel * Genome size estimates are baseD on the k-mer frequency Distribution of Illumina reaDs. missing SCOs, indicating gaps in the draft wAlbB M. ozzardi, and their Wolbachia endosymbionts. Genome size estimates baseD on other filarial Wolbachia anD wCle assembly. wFol Mansonella Genomes: Challenges and Solutions Completeness of Mansonella Genomes %BUSCOs • High levels of human DNA in (up to 95%) as they are derived from patient samples. Work in Progress • The completeness of gene content of assembled draft genomes was evaluated using ü Reduce human DNA using NEBNext® Microbiome DNA Enrichment Kit the BUSCO software (Simão et al. 2015). • Refinement and improvement of all the assembled genomes and annotations. ü Sequence as deep as possible • Intra and inter-species comparisons of genomes. ü Bioinformatic filtering of reads and contigs originating from human DNA • The BUSCO score of a genome is • Metabolic pathway analysis for understanding the symbiotic relationship of calculated as the percentage of • Limited amounts of parasite material. M. perstans Mansonella and their Wolbachia. ü universal Single Copy Orthologs (SCOs) Illumina library preparation using NEBNext® Ultra™ II DNA Library Prep Kit M. ozzardi encoded by a genome, as compared to Conclusions B. malayi Sample from Cameroon a baseline set of SCOs known to be Illumina Sequencing • Draft assemblies of M. perstans and M. ozzardi have been obtained. Mp1 gDNA < 10 ng highly conserved in a particular taxa. Loa loa Mansonella perstans • Draft genomes for filarial Wolbachia wMpe and wMoz have been obtained. These PacBio Sequencing Mp2 Sample from: Cameroon O. volvulus represent the first ever genomic data from a nematode Wolbachia of supergroup F. 10X Genomics gDNA = 800 ng • M. perstans and M. ozzardi encode for Illumina Sequencing W. bancrofti • Intra- and inter-species comparisons of genomes will enable investigations and more than 85% of the 982 SCOs that provide insights into the biology and evolution of some of the most highly neglected are conserved across all nematodes. %BUSCOs Sample from Brazil filarial parasites. Illumina Sequencing Mo1 gDNA < 10 ng References: Mansonella ozzardi • Their BUSCO score is similar to that from other sequenced filarial genomes, • Lefoulon, E. et al. (2016) Breakdown of coevolution between symbiotic bacteria Wolbachia and their filarial hosts. PeerJ 4, e1840. Sample from Venezuela • Lima, N. F. et al. (2016) Mansonella ozzardi: a neglected New World filarial nematode. Pathogens and Global Health 110. Mo2 Illumina Sequencing suggesting that the Mansonella draft assemblies contain most of the protein coding gDNA < 10 ng • Simão, F. A. et al. (2015) BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31. genes they are estimated to encode for. • Simonsen, P. E. et al. (2011) Mansonella perstans filariasis in Africa. Acta Tropica 120. This work was funded by New England Biolabs.
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