Chromosome-Level Genome Sequence of Leishmania (Leishmania) tropica Strain CDC216-162, Isolated from an Afghanistan Clinical Case.

PacBio and Illumina MiSeq platforms were used for genomic sequencing of a Leishmania (Leishmania) tropica strain isolated from a patient infected in Pakistan. PacBio assemblies were generated using Flye v2.4 and polished with MiSeq data. The results represent a considerable improvement of the currently available genome sequences in the GenBank database.

ANNOUNCEMENT

Leishmaniasis is a vector-born disease caused by >20 species of parasites in the genus Leishmania which affects millions of people worldwide and causes thousands of deaths yearly (1, 2). The spectrum of the disease includes the following three main clinical manifestations, depending on the Leishmania species: (i) cutaneous leishmaniasis (CL), the most common clinical manifestation, characterized by development of ulcerative cutaneous lesions; (ii) mucocutaneous leishmaniasis (MCL), which can cause permanent disfiguration of oral and nasopharyngeal mucosa or death; and (iii) visceral leishmaniasis (VL) that is fatal in most untreated cases (3–5). Recently, genome sequence data have been used to determine phylogenetic relationships between Leishmania parasites, providing insights into parasite classification, genetic polymorphism, virulence, and drug resistance and also supporting the development of specific diagnostics for preventing the morbidity and mortality of the disease (17–20).

In this study, we present a chromosome-level genome sequence of Leishmania (Leishmania) tropica, an etiological agent of CL endemic in the Middle East, northern Africa at the Mediterranean Sea, and some parts of Asia. A skin biopsy specimen obtained from an American traveler in Afghanistan infected with Leishmania (L.) tropica was cultured in 10% fetal bovine serum (FBS)-RPMI axenic medium (Life Technologies, CA). PacBio and Illumina MiSeq libraries were prepared with DNA from cultured parasites using the MagAttract high-molecular-weight (HMW) DNA kit (Qiagen, MD) as previously described (6–8). PacBio libraries were prepared using the PacBio 20-kb kit (Pacific Biosciences, CA), size selected with BluePippin (Sage Science, MA), and sequenced with C4v2 chemistry for 360-minute movies on the RS II instrument (Pacific Biosciences). Dual-indexed libraries were prepared with the NEBNext Ultra library prep kit (New England BioLabs, MA) and sequenced using the MiSeq 2 × 250-cycle sequencing kit (Illumina, CA). PacBio filtered reads (minlength = 1,000; number of reads = 293,219; average length = 9,974) were de novo assembled using Flye v2.4 (9–13) (-g 32m).

The de novo assembly comprised 88 fragments which were scaffolded using Companion v1.0.1 (14) (default parameters), using Leishmania (L.) major as the reference genome. The resultant scaffolds were examined for correct orderings and orientation using Mauve, and the misassembled contigs were manually curated and fixed. To improve the quality and contiguity of scaffolds, PacBio reads were mapped to chromosome scaffolds generated by Companion, mapped reads were binned by chromosomes, and each bin was assembled independently using Flye v 2.4 (-g 2m), resulting in 36 scaffolds. These scaffolds were processed again using Companion as described above, producing 36 contigs representing each of the expected 36 chromosomes. Additionally, one round of polishing with Illumina reads was performed using POLCA (15) to correct inherent PacBio sequencing errors. The final assembly was evaluated for completeness by BUSCO 4.0.6 (16) against euglenozoa_odb10 (completeness [C], 100.0%; complete and duplicated [D], 0.0%; complete and single-copy [S], 100.0%; fragmented [F], 0.0%; missing [M], 0.0%; number of genes used [n], 130). Default software parameters were used in this study, except where otherwise noted.

High-quality contiguous genomic sequences are crucial for more robust background and investigations in the leishmaniasis field, which are essential for the development of more species-specific diagnostic tests and clinical management. Therefore, as shown in Table 1, the proposed assembly with fully resolved Leishmania (L.) tropica chromosomes presented here represents a considerable improvement over the existing assemblies.

TABLE 1

Comparison of proposed assemblies of Leishmania (L.) tropica

L. (L.) tropica isolate	Read type	Total genome length (bp)	No. of scaffolds	No. of contigs	N50 length (bp)	L50 count	G+C content (%)	Coverage (×)	GenBank accession no.
L590	Illumina MiSeq	32,989,014	448	1,938	32,739	275	59.30	253	GCA_000410715.1
ATCC 50129	Illumina MiSeq	30,870,161	1,928	1,928	32,161	274	59.90	150	GCA_011316065.1
WHO2017-IKa	Illumina MiSeq	32,139,927	9,499	20,898	3,299	20,898	57.20	150	GCA_003067545.1
WHO2015-IKb	Illumina HiSeq	32,280,712	17,013	18,935	6,327	1,404	59.60	150	GCA_003352575.1
CDC216-162	PacBio, Illumina MiSeq	32,702,166	36 + bin	36 + bin	1,070,514	11	59.61	75 (PacBio RS II CLR), 100 (Illumina)	GCA_014139745.1

MHOM/LB/2017/IK.

MHOM/LB/2015/IK.

Data availability.

This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JACCHM000000000 and under BioProject number PRJNA484340. The version described in this paper is the first version, JACCHM010000000. The accession numbers for raw reads are SRR7867286, SRR7867287, SRR7867288, SRR7867289, SRR7867290, SRR7867291, and SRR10771526.