First Draft Genome Sequence of Leishmania (Viannia) lainsoni Strain 216-34, Isolated from a Peruvian Clinical Case.

We present here the first draft genome sequence of Leishmania (Viannia) lainsoni strain 216-34, sequenced using PacBio and MiSeq platforms. PacBio contigs were generated from de novo assemblies using CANU version 1.6 and polished using Illumina reads.

ANNOUNCEMENT

In South and Central America, cutaneous leishmaniasis (CL) is caused by several Leishmania species in the Leishmania and Viannia subgenera (1, 2). Patients infected with some Leishmania (Viannia) strains of L. (V.) braziliensis, L. (V.) guyanensis, and L. (V.) panamensis can also develop mucocutaneous leishmaniasis (MCL), a mucosa-destructive sequela affecting the naso-oropharyngeal and laryngeal tracts (3). Other species in the Viannia subgenus, such as L. (V.) lainsoni and L. (V.) peruviana, are associated only with CL. Treatment is recommended for most cases of CL. However, the drugs typically used for treating CL are associated with several adverse side effects. In addition, the choice of therapeutic agents and their efficacy vary by clinical characteristics and parasite-specific factors (3 – 5). Therefore, DNA-based diagnostic tests that allow specific identification of infecting parasite species are critical for appropriate clinical management, including decisions regarding which treatment is indicated and how to monitor for relapse and sequelae.

As a part of our efforts to improve the quality of species-specific diagnostics for leishmaniasis, we present here the first draft genome sequence of L. (V.) lainsoni, that of strain 216-34, derived from a parasite isolated in our laboratory from a clinical case from Peru in 2016. Parasites grown in RPMI 1640 axenic medium (Life Technologies, CA) were initially identified on the basis of morphological analysis (6). Later, the species identity of L. (V.) lainsoni was confirmed using multilocus sequence analysis (MLSA) targeting glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICD), malate dehydrogenase (MDH), and mannose-6-phosphate isomerase (MPI) Leishmania sp. enzyme-coding genes. The sequencing data, generated on an ABI 3130xl DNA analyzer (Applied Biosystems, CA), were compared with the NCBI nucleotide database using the BLASTn algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch).

MiSeq and PacBio libraries were prepared with genomic DNA purified using the MagAttract high-molecular-weight (HMW) DNA kit (Qiagen, MD). For the short reads, libraries were prepared with NEBNext Ultra library prep reagents (New England BioLabs, MA), barcoded with indices produced at the CDC Biotechnology Core Facility, and sequenced using a MiSeq 2 × 250-cycle sequencing kit (Illumina, CA). Sequence reads were filtered for read quality, base called, and demultiplexed using bcl2fastq (version 2.19). Long-read sequencing data were obtained from a 20-kb DNA library and prepared using standard PacBio 20-kb library protocols (Pacific Biosciences, CA). The library was size selected with the BluePippin system (Sage Science, MA), bound to polymerase using the DNA/polymerase binding kit P6v2, loaded onto 6 single-molecule real-time (SMRT) cells, and sequenced with C4v2 chemistry for 360-min movies on the RS II instrument (Pacific Biosciences, CA).

The reads (246,178 reads; mean read length, 12,377 bp) from the RS II instrument were filtered and de novo assembled using Canu version 1.6 (7) with default parameters. Non-Leishmania contigs identified using the NCBI-BLASTn algorithm were removed, and the remaining contigs were polished and corrected using unicycler_polish (Unicycler package version 4.4) (8) with the 26,942,306 Illumina reads. The L. (V.) lainsoni genome assembly comprises 140 contigs covering 34,156,530 bp, with an L50 value of 17, N50 value of 638,860 bp, G+C content of 57.83%, and average read coverage of 75×.

The first draft genome sequence of L. (V.) lainsoni presented here is an important resource for diagnostic applications and research on the Leishmania genus.

Data availability.

The draft genome contigs of L. (V.) lainsoni 216-34 have been deposited in GenBank under accession number RAIA00000000 and in the SRA under accession numbers SRR7883038, SRR7883039, SRR7883040, and SRR8179821 (BioProject number PRJNA484340).