Draft Genome Sequences of Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) mexicana, and Leishmania (Leishmania) aethiopica, Potential Etiological Agents of Diffuse Cutaneous Leishmaniasis

We present here the draft genome sequences of Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) mexicana, and Leishmania (Leishmania) aethiopica, potential etiological agents of diffuse cutaneous leishmaniasis (DCL). Sequence data were obtained using PacBio and MiSeq platforms.

ABSTRACT

We present here the draft genome sequences of Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) mexicana, and Leishmania (Leishmania) aethiopica, potential etiological agents of diffuse cutaneous leishmaniasis (DCL). Sequence data were obtained using PacBio and MiSeq platforms. The PacBio assemblies generated using Canu v1.6 are more contiguous than are those in the available data.

ANNOUNCEMENT

Leishmaniasis is a vector-borne disease caused by more than 20 species of parasites in the Viannia and Leishmania subgenera, affecting millions of people yearly (1, 2). Cutaneous leishmaniasis (CL) is the most prevalent clinical manifestation, which usually heals spontaneously within 6 months to 3 years. Diffuse CL (DCL) is a rare and poorly understood form of CL, characterized by non-self-healing chronic lesions in large areas of the skin, with high parasite proliferation and resistance to most therapeutic agents (3–5). DCL is caused mostly by strains of Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) mexicana in Latin America and Leishmania (Leishmania) aethiopica in the Horn of Africa. Treatment of the primary CL is required to prevent the morbidity and mortality of leishmaniasis, but the choice and effectiveness of the therapy depend on the parasite species and the host immune response (1, 5–7). Therefore, species-specific diagnostic methods are crucial for clinical management.

To increase the quality of Leishmania species reference sequences, we submitted to the database the draft genome sequences of the species L. (L.) amazonensis, L. (L.) mexicana, and L. (L.) aethiopica. The parasites listed in Table 1 were identified as infecting species of three CL clinical cases from French Guyana, Texas, and Ethiopia, respectively, using the CDC’s current diagnostic approach (8, 9). A portion of each CL specimen was cultivated at 26°C in 20 ml RPMI medium (Invitrogen, GA) enriched with 0.5% HEPES, 0.25% l-glutamine, 0.02% folic acid, 15% fetal bovine serum, 0.02% hemin, 1% minimal essential medium (MEM) vitamin solution (100×), penicillin-streptomycin (75 I/ml to 75 μg/ml), and gentamicin (50 μg/ml) (pH 7.4). When the parasites reached log phase, 1.5-ml aliquots of the culture were preserved with 10% dimethyl sulfoxide (DMSO) in liquid nitrogen. For this study, parasites were reactivated by mixing the thawed culture aliquots with 20 ml of fresh RPMI medium. After 2 to 3 weeks of incubation, 1.0 ml of culture aliquots was centrifuged at 6,000 × g for 15 min, and the pellet was used for DNA extraction using the MagAttract high-molecular-weight (HMW) DNA kit (Qiagen, MD).

TABLE 1.

Assembly metrics for L. (L.) amazonensis, L. (L.) mexicana, and L. (L.) aethiopica and their corresponding reference strains

Strain	Read type(s)	Total length (bp)	No. of scaffolds	No. of contigs	N50 length (bp)	L50	Longest contig (bp)	No. of Illumina reads	No. of PacBio readsa	G+C content (%)	Coverage (×)
L. (L.) amazonensis CDC210-L1346	PacBio, Illumina	33,504,997	92	92	850,106	12	3,425,950	30,427,462	262,667	59.71	75
L. (L.) amazonensis M2269b	454, Illumina	29,029,348	2,627	2,944	19,306	430	113,027	NA	NA	59.26	NA
L. (L.) mexicana CDC215-L49	PacBio, Illumina	32,057,209	55	55	825,953	11	3,409,057	30,816,844	323,584	59.79	87
L. (L.) mexicana U1103b	Sanger, Illumina	32,108,741	588	927	164,930	56	1,024,353	NA	NA	59.8	NA
L. (L.) aethiopica CDC209-L1204	PacBio, Illumina	33,648,436	118	118	763,733	15	1,670,940	10,767,274	245,491	60.38	74
L. (L.) aethiopica L147b	Illumina	31,630,816	160	1,698	39,581	240	168,996	NA	NA	60.07	NA

^aNA, not available.

^bIndicates currently available assemblies in the public databases.

The DNA extracts were used for the preparation of Illumina MiSeq and PacBio libraries. Dual-indexed libraries using NEBNext Ultra library prep (New England BioLabs, MA) and barcoding indices were manufactured at the CDC-Biotechnology Core Facility. These libraries were sequenced on a MiSeq platform (Illumina, CA) using the MiSeq 2 × 250-cycle sequencing kit. Sequence reads were filtered, base called, and demultiplexed using bcl2fastq (v2.19), with default parameters. PacBio libraries were prepared using the standard PacBio 20-kb procedure (Pacific Biosciences, CA). The libraries were size selected using BluePippin (Sage Science, MA), bound to the polymerase using the DNA/polymerase binding kit P6 v2, loaded onto 3 to 5 single-molecule real-time (SMRT) cells, and sequenced with C4 v2 chemistry for 360-min movies on the RS II instrument (Pacific Biosciences).

The filtered PacBio (minlength = 1,000) reads were de novo assembled using Canu v1.6 (genomeSize = 32m) (10). After removing contigs with a coverage of <10×, the remaining contigs were corrected with Illumina reads using unicycler_polish (default parameters; Unicycler package v4.4) (11). As shown in Table 1 , the resultant assemblies are more contiguous than are those in the data available in GenBank for L. (L.) amazonensis LeiAma1.0 (GCA_000438535) (12), L. (L.) mexicana ASM23466v4 (GCA_000234665) (13), and L. (L.) aethiopica L147 (GCA_000444285).

Data availability.

The genome contigs have been deposited in GenBank under accession numbers RZOD00000000 [L. (L.) amazonensis CDC210-L1346], RZOC00000000 [L. (L.) mexicana CDC215-L49], and RZOE00000000 [L. (L.) aethiopica CDC209-L1204] under BioProject number PRJNA484340. The accession numbers for the raw reads are SRR8377732, SRR7867275, SRR7867276, SRR7867277, SRR8377734, SRR7867272, SRR7867273, SRR7867285, SRR7867284, SRR8377733, SRR7867278, SRR7867279, SRR7867280, SRR7867281, and SRR7867274.