Resequencing of the Leishmania infantum (strain JPCM5) genome and de novo assembly into 36 contigs

Abstract

Leishmania parasites are the causative of leishmaniasis, a group of potentially fatal human diseases. Control strategies for leishmaniasis can be enhanced by genome based investigations. The publication in 2005 of the Leishmania major genome sequence, and two years later the genomes for the species Leishmania braziliensis and Leishmania infantum were major milestones. Since then, the L. infantum genome, although highly fragmented and incomplete, has been used widely as the reference genome to address whole transcriptomics and proteomics studies. Here, we report the sequencing of the L. infantum genome by two NGS methodologies and, as a result, the complete genome assembly on 36 contigs (chromosomes). Regarding the present L. infantum genome-draft, 495 new genes have been annotated, a hundred have been corrected and 75 previous annotated genes have been discontinued. These changes are not only the result of an increase in the genome size, but a significant contribution derives from the existence of a large number of incorrectly assembled regions in current chromosomal scaffolds. Furthermore, an improved assembly of tandemly repeated genes has been obtained. All these analyses support that the de novo assembled L. infantum genome represents a robust assembly and should replace the currently available in the databases.

Figure 1

Schematic overview of the workflow leading to the L. infantum genome assembly. Input files (Raw Reads) are represented as yellow rhomboids. All the different software and processes are shown in blue boxes. Output files are represented in green rhomboids. Discarded data are shown in red rhomboids. See Materials and Methods section for additional details.

Figure 2

Read-depth analysis along the chromosomes formed by the fusion of two PacBio-assembled contigs. Coverage was determined by sliding window analysis (bin 200 pb) with either Illumina (in blue) or PacBio (in red) reads, along chromosomes 7, 12, 15, 22, 26, 28, 33 and 35. The sizes of the contigs are shown by lines with arrow-heads. Chromosomes 7 (panel A) and 35 (panel H) were joined by the SSPACE-standard tool. Chromosomes 12, 15, 22, 26 and 28 (panels B–F) were joined using the minimus 2 assembler. Finally, chromosome 33 (panel G) was joined by the SSPACE-LongRead tool.

Figure 3

Gene copy number in the beta-tubulin locus at the chromosome 33. Panel (A): Genomic structure of the region containing the beta-tubulin locus in the L. major Friedlin genome (LmjF), in the L. infantum genome assembled in this work (LinJ-New) and in the current L. infantum assembly (LinJ-Ref).The identity percentage of the BLAST alignment (using the tab –format output of BLAST) is shown by shading with brown hue (scale at top left ranges from 90 to 100% of sequence identity). (B) Distribution of L. major Illumina reads (unpublished laboratory data) along the beta-tubulin locus in the L. major current genome (GeneDB.org). (C) Distribution of L. infantum sequence-reads (Illumina in blue and PacBio in red) along the beta-tubulin genomic region using as reference the L. infantum genome assembled in this work. (D) Distribution of L. infantum reads (Illumina in blue and PacBio in red) along the region containing the beta-tubulin locus in current L. infantum genome (version 9; Tritryp.org).

Figure 4

Gene copy number in the glucose transporter 2 locus. (A) Genomic structure of the region containing the glucose transporter 2 locus in the L. major Friedlin genome (LmjF), in the L. infantum genome assembled in this work (LinJ-New) and in the current L. infantum assembly (LinJ-Ref). See legend to Fig. 3 for the meaning of color codes. (B) Distribution of L. major Illumina reads along the glucose transporter 2 region in the L. major current genome (GeneDB). (C) Distribution of L. infantum sequence-reads (Illumina in blue and PacBio in red) along the glucose transporter 2 genomic region using as reference the L. infantum genome assembled in this work. (D) Distribution of L. infantum reads (Illumina in blue and PacBio in red) along the region containing the glucose transporter 2 locus in current L. infantum genome (version 9; Tritryp.org).

Figure 5

Schematic illustration of mis-assembled regions in the current L. infantum genome. Synteny blocks, represented by different colors, in chromosome 7 (panel A) and chromosome 13 (panel B) after pair-wise comparisons between the L. major Friedlin genome (top), the L. infantum newly assembled genome (middle) and L. infantum reference genome (bottom). Pairwise alignments were generated by the progressive MAUVE algorithm, which uses color codes to depict blocks of conserved regions. Sections located underneath the x-axis show inversion events.