Nearly Complete Genome Sequence of Brugia malayi Strain FR3

Lymphatic filariasis affects ∼120 million people and can result in elephantiasis and hydrocele. Here, we report the nearly complete genome sequence of the best-studied causative agent of lymphatic filariasis, Brugia malayi. The assembly contains four autosomes, an X chromosome, and only eight gaps but lacks a contiguous sequence for the known Y chromosome.

B rugia malayi is a causative agent of lymphatic filariasis, which affects ϳ120 million people and can result in elephantiasis and hydrocele. An ϳ71-Mbp B. malayi draft genome sequence was previously produced using Sanger sequencing with 8,180 scaffolds and an N 50 value of ϳ93 kbp (1,2). Here, we used long-read sequencing and manually curated optical maps to complete this B. malayi genome.
B. malayi adult worms were obtained directly from the filarial nematode repositories at TRS Labs and the Filariasis Research Reagent Resource Center (FR3) (3)-the two major repositories that both independently maintain the same lineage of B. malayi originally from a green leaf monkey (4). High-molecular-weight genomic DNA was prepared by grinding frozen worms in liquid nitrogen and transferring them to 100 mM Tris-HCl (pH 8.5), 50 mM NaCl, 50 mM EDTA, 1% SDS, 1.1% ␤-mercaptoethanol, and 100 g/ml NEB proteinase K at 55°C for 4 h with rocking. DNA was spooled from an ethanol precipitation following a phenol-chloroform extraction. DNA was suspended in Tris-EDTA (TE) (pH 8.0) with 25 g/ml Epicentre RNase A at 37°C for 1 h followed by phenol-chloroform extraction, precipitation, and centrifugation at 12,000 ϫ g at 4°C. Genomic DNA (30 g) was sheared to 20 kbp with a Covaris g-TUBE. A 7-kbp Sage Science Blue Pippin size-selected SMRTbell library was prepared, and 11.3 Gbp of sequence data (3,922,808 reads; N 50 , 17,971 bp) were produced on a Pacific Biosciences RS II instrument (P5-C3; 180 min).
For optical mapping, individual phosphate-buffered saline (PBS)-washed B. malayi male worms were placed into ϳ50-l plugs of 1% InCert agarose (Lonza, Rockland, ME) in PBS that were extruded into 1 ml of 50°C 1% (wt/vol) N-lauroylsarcosine, 2 mg/ml proteinase K, and 0.5 M EDTA (pH 9.5) and incubated overnight with rocking at 50°C. Plugs were washed 5 times for 1 h each in TE (pH 8.0), with rocking at 4°C, and then stored at 4°C in 0.5 M EDTA (pH 8.0). Stretched and immobilized DNA was digested with NEB SpeI and AflII separately and fluorescently stained, generating ϳ80ϫ optical data depth. An OpGen Argus optical mapping system (2015 version), with proprietary MapManager (2015 version) and MapSolver version 3.1 software, resolved a 96.58-Mbp B. malayi SpeI optical map of 17 contigs and a 77.57-Mbp AflII optical map of 12 contigs.
The resulting 87-Mbp assembly of 196 scaffolds has a GC content of 28% and an N 50 value of 14.2 Mbp with 4 autosomes and an X chromosome but is lacking a contiguous Y chromosome despite numerous efforts to assemble it.

ACKNOWLEDGMENTS
This project was funded in part by the Burroughs-Wellcome Fund to E.G., federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19AI110820 to J.C.D.H., J.M.F., and M.L.M., and core support from Wellcome (grants WT098051 and WT206194) to the Wellcome Sanger Institute and Medical Research Council (UK) funding to M.B. and M.P. (grant MR/L001020/1).
We thank Karen Brooks for help with manual finishing.