ABSTRACT
We report the high-quality draft assemblies and gene annotations for 13 species and/or strains of the protozoan parasite genera Leishmania, Endotrypanum, and Crithidia, which span the phylogenetic diversity of the subfamily Leishmaniinae within the kinetoplastid order of the phylum Euglenazoa. These resources will support studies on the origins of parasitism.
ANNOUNCEMENT
Leishmania species are widespread parasites of mammals transmitted by biting insects. Over 1.7 billion people worldwide are at risk, with hundreds of millions of people infected (1–4). The genus comprises more than 50 species, which are primarily zoonotic but in humans cause disease ranging from mild cutaneous lesions to more disseminated forms to fatal visceralizing disease (5). While parasitism by Leishmania has been intensively studied, the species-specific factors that enable mammalian or insect host infections are less well understood. To provide a broad phylogenetic snapshot, we selected a spectrum of species and strains across the subfamily Leishmaniinae (5), targeting lineages within the subgenera Leishmania, Viannia, and Mundinia, as well as the allied genus Endotrypanum and the outgroup Crithidia fasciculata (Table 1). The WU Institutional Biosafety Committee reviewed and approved the parasite work reported here (01-015).
TABLE 1.
Description of Leishmania species and strains, including assembly parameters and links
| Genus or subgenus | Species | Strain | WHO code | Source | Provenance | BioProject accession no.a | Sequencing platforms | Assembler | No. of contigs | N50 (bp) | Assembly size (bp) | % GC | No. of pseudochromosomes | No. of protein-coding genes | Reference for other assembly |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| L. Leishmania | L. major | SD75.1 (clone) | MHOM/SN/74/SD | Human, cutaneous | D. Sacks, Bethesda, MD | PRJNA50303 | 454, Illumina | Newbler | 891 | 95,380 | 31,727,271 | 59.3 | 36 | 8,818 | |
| LV39 (clone 5) | MHOM/Sv/59/P | Human, cutaneous | R. Titus, Boston, MA | PRJNA50301 | 454, Illumina | Newbler | 1,667 | 71,452 | 31,961,985 | 59.5 | 36 | 8,971 | |||
| L. gerbilli | LEM452 | MRHO/CN/60/GERBILLI | Gerbil | P. Bastien, J.-P. Dedet, Montpellier, France | PRJNA192717 | 454, Illumina | AllPaths | 1,248 | 57,008 | 30,822,621 | 59.6 | 36 | 8,599 | ||
| L. turanica | LEM423 | MRHO/SU/65/VL | Gerbil | P. Bastien, J.-P. Dedet, Montpellier, France | PRJNA192712 | 454, Illumina | AllPaths | 1,669 | 39,210 | 30,876,294 | 59.5 | 36 | 8,608 | ||
| L. arabica | LEM1108 | MPSA/SA/83/JISH220 | Psammomys sp. | P. Bastien, J.-P. Dedet, Montpellier, France | PRJNA192710 | 454, Illumina | AllPaths | 1,530 | 52,119 | 30,774,332 | 59.2 | 36 | 8,646 | ||
| L. tropica | L590 | MHOM/IL/1990/P283 | Human, cutaneous | C. Jaffe, Jerusalem, Israel | PRJNA169676 | 454, Illumina | AllPaths | 1,938 | 32,739 | 31,326,083 | 59.6 | 36 | 8,824 | 12 | |
| L. aethiopica | L147 | MHOM/ET/1972/L100 | Human, diffuse cutaneous, relapsing | C. Jaffe, Jerusalem, Israel | PRJNA169673 | 454, Illumina | AllPaths | 1,758 | 38,498 | 31,026,739 | 60.1 | 36 | 8,722 | ||
| L. Viannia | L. braziliensis | M2903 | MHOM/BR/75/M2903 | Human, cutaneous | J. Shaw, Brazil | PRJNA165955 | 454, Illumina | Newbler | 3,934 | 61,918 | 32,590,753 | 57.4 | 35 | 9,269 | |
| L. panamensis | L13 | MHOM/COL/81/L13 | Human, mucosal | N. Saravia, Cali, Colombia | PRJNA165959 | 454, Illumina | AllPaths | 3,163 | 22,576 | 31,108,242 | 57.4 | 35 | 8,665 | ||
| L. Mundinia | L. enriettii | LEM3045 | MCAV/BR/95/CUR3 | Cavia porcellus (guinea pig) | P. Bastien, J.-P. Dedet, Montpellier, France | PRJNA192711 | 454, Illumina | AllPaths | 1,171 | 102,666 | 30,427,298 | 59.3 | 36 | 8,731 | 14 |
| L. martiniquensis | LEM2494 | MHOM/MQ/92/MAR1 | Human, diffuse cutaneous, HIV | P. Bastien, J.-P. Dedet, Montpellier, France | PRJNA192703 | 454, Illumina | AllPaths | 628 | 147,290 | 30,528,357 | 59.6 | 36 | 8,483 | 14 | |
| Endotrypanum | Endotrypanum monterogei | LV88 | None | Choloepus hoffmanni (sloth) | Michael Chance, Liverpool, UK | PRJNA165953 | 454, Illumina | Newbler | 3,517 | 33,059 | 32,086,870 | 52.5 | 36 | 8,285 | 13 |
| Crithidia | Crithidia fasciculata | Cf-C1 (clone) | None | Mosquito | Larry Simpson, Los Angeles, CA | PRJNA165885 | 454, Illumina, PacBio | HGAP | 494 | 778,443 | 41,297,378 | 57.0 | 30 | 9,619 | 8 |
Each BioProject link contains links to the current assembly, primary data sets, and other relevant information.
Parasites were cultivated in M199 or Schneider’s medium (6) and grown to late log phase before harvesting, lysis, and DNA purification by phenol-chloroform extraction and/or banding in CsCl gradients (to remove mitochondrial maxi- or minicircle DNA). Sequencing libraries were generated using the Illumina paired-end DNA sample preparation kit (PE-102-1001) according to the manufacturer’s directions. Fragment libraries of 3 and 8 kb were prepared using protocols for 454 sequencing (Roche Life Sciences). Sequencing was performed on either a 454 GS FLX Titanium (average read length, 305 bp; Roche 454 Life Sciences) or Illumina Genome Analyzer IIx (GAIIx) and HiSeq 2000 instruments (paired-end 100-bp format), except for Crithidia, which additionally utilized long reads generated on an RS II instrument (P5/C3 chemistry; Pacific Biosciences) (7). The total sequence genome coverage on the Illumina GAIIx and HiSeq 2000 instruments was on average 105× with tiered library insert sizes (50× fragments; 45 × 3 kb, 10 × 8 kb, and 0.05 × 40 kb). For all Illumina sequences, we used the read processing steps within the AllPaths-LG (8) software prior to de novo assembly, which incorporates read error correction methods described by Pevzner et al. (9). Genome assemblies were conducted with default parameters using Newbler v2.0.1 (10) for 454 reads, AllPaths-LG (8) for Illumina reads, and HGAP v3 (11) for long reads (Table 1). Contigs and scaffolds were organized into pseudochromosomes using ABACAS2 (https://github.com/satta/ABACAS2), a successor to ABACAS (12), by alignment with the Leishmania major Friedlin genome sequence, with the exception of Leishmania braziliensis M2903 and Leishmania panamensis, which were aligned to the L. braziliensis M2904 genome. The estimated haploid genome sizes ranged from 30.4 to 41.3 megabases (13).
Gene annotations were performed using the comprehensive Companion tool, which incorporates a variety of de novo prediction criteria, as well as information from closely related genomes when available (14). The number of protein-coding genes predicted ranged from 8,285 to 9,619, typical of other Leishmania species (13). Full annotations, as well as a variety of tools for the visualization or analysis of these genomes, are available from TriTrypDB (www.tritrypdb.org).
Data availability.
The assemblies have been deposited in the NCBI GenBank repository under the BioProject accession numbers in Table 1, including links to the primary data and annotations (PRJNA50303, PRJNA50301, PRJNA192717, PRJNA192712, PRJNA192710, PRJNA169676, PRJNA169673, PRJNA165955, PRJNA165959, PRJNA192711, PRJNA192703, PRJNA165953, and PRJNA165885). The chromosome builds are available through the TriTrypDB portal (http://tritrypdb.org/tritrypdb).
ACKNOWLEDGMENTS
NIH grant AI29646 to S.M.B. provided support for N.S.A., D.E.D., and L.-F.L.; NIH-NHGRI grant HG00307907 to R.K.W. supported W.C.W. and C.T.; and AI103858 to P.J.M. supported G.R. Funding to C.H.-F. from Wellcome Trust grants WT099198MA and WT108443MA provided support for F.S.-F., A.S., and S.S.
We thank Patrick Minx for assembly curation. We thank the colleagues listed in Table 1 for generously providing the strains and Brian Brunk, David Roos, Thomas Otto, and Matt Berriman for discussions.
This research was funded in whole, or in part, by the Wellcome Trust (WT099198MA and WT108443MA). For the purpose of open access, the author has applied a CC BY public copyright license to any author accepted manuscript version arising from this submission.
Contributor Information
Stephen M. Beverley, Email: stephen.beverley@wustl.edu.
Vincent Bruno, University of Maryland School of Medicine.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The assemblies have been deposited in the NCBI GenBank repository under the BioProject accession numbers in Table 1, including links to the primary data and annotations (PRJNA50303, PRJNA50301, PRJNA192717, PRJNA192712, PRJNA192710, PRJNA169676, PRJNA169673, PRJNA165955, PRJNA165959, PRJNA192711, PRJNA192703, PRJNA165953, and PRJNA165885). The chromosome builds are available through the TriTrypDB portal (http://tritrypdb.org/tritrypdb).