Long-Read Sequencing and De Novo Genome Assembly Pipeline of Two Plasmodium falciparum Clones (Pf 3D7, Pf W2) Using Only the PromethION Sequencer from Oxford Nanopore Technologies without Whole-Genome Amplification

Abstract

Antimalarial drug resistance has become a real public health problem despite WHO measures. New sequencing technologies make it possible to investigate genomic variations associated with resistant phenotypes at the genome-wide scale. Based on the use of hemisynthetic nanopores, the PromethION technology from Oxford Nanopore Technologies can produce long-read sequences, in contrast to previous short-read technologies used as the gold standard to sequence Plasmodium. Two clones of P. falciparum (Pf3D7 and PfW2) were sequenced in long-read using the PromethION sequencer from Oxford Nanopore Technologies without genomic amplification. This made it possible to create a processing analysis pipeline for human Plasmodium with ONT Fastq only. De novo assembly revealed N50 lengths of 18,488 kb and 17,502 kb for the Pf3D7 and PfW2, respectively. The genome size was estimated at 23,235,407 base pairs for the Pf3D7 clone and 21,712,038 base pairs for the PfW2 clone. The average genome coverage depth was estimated at 787X and 653X for the Pf3D7 and PfW2 clones, respectively. This study proposes an assembly processing pipeline for the human Plasmodium genome using software adapted to large ONT data and the high AT percentage of Plasmodium. This search provides all the parameters which were optimized for use with the software selected in the pipeline.

Keywords: Pf3D7; PfW2; Plasmodium falciparum; PromethION; genome assembly; long-read sequencing; nanopore.

Figure 1

Assembly processing pipeline of Plasmodium falciparum Oxford Nanopore long-read sequencing. (Bash script is in Supplementary File S1). Raw assembly (Sky blue); optional, non automized assembly optimisation step (Grey); refinement processing (Green): the step should be processed as long as it reduces the reported mutation number (a repetition corresponds to following the green dotted arrow pathway). Quality control (Orange): to help set the parameters and thresholds. Optional skeleton build (Navy Blue): if no satisfying reference exists for your strain.

Figure 2

Sequencing depth of Plasmodium falciparum 3D7 clone genome assembly for the 14 chromosomes and the apicoplast and the mitochondria. The figure was realized with Samtools depth and R script (in Supplementary File S2). The mean depth per chromosomes were as follows: 3D7_chromosome_1 (754.4 ± 64.6); 3D7_chromosome_2 (764 ± 95.5); 3D7_chromosome_3 (768.3 ± 56.3); 3D7_chromosome_4 (808.9 ± 161.5); 3D7_chromosome_5 (774 ± 81.4); 3D7_chromosome_6 (784 ± 81.7); 3D7_chromosome_7 (779.7 ± 59.8); 3D7_chromosome_8 (762 ± 68.1); 3D7_chromosome_9 (765 ± 59.8); 3D7_chromosome_10 (760.8 ± 56.2); 3D7_chromosome_11 (769.6 ± 73.7); 3D7_chromosome_12 (774.8 ± 109.7); 3D7_chromosome_13 (779.7 ± 126); 3D7_chromosome_14 (776.9 ± 91.4); 3D7_apicoplaste (465.8 ± 113.4); and 3D7_mitochondria (7922.1 ± 151.9).

Figure 3

Sequencing depth of Plasmodium falciparum W2 clone genome assembly for the 14 chromosomes and the apicoplast and the mitochondria. The figure was realized with Samtools depth and R script (in Supplementary File S2). The mean depths per chromosome were as follows: PfW2_chromosome_1 (946 ± 1041.9); PfW2_chromosome_2 (731 ± 487.8); PfW2_chromosome_3 (669.7 ± 381); PfW2_chromosome_4 (749.4 ± 545.6); PfW2_chromosome_5 (705.9 ± 264.2); PfW2_chromosome_6 (669.7 ± 223.7); PfW2_chromosome_7 (685.7± 366.4); PfW2_chromosome_8 (660.6 ± 334.2); PfW2_chromosome_9 (632.8 ± 144.6); PfW2_chromosome_10 (627.5 ± 62.8); PfW2_chromosome_11 (620.9 ± 59.5); PfW2_chromosome_12 (635.9 ± 123.6); PfW2_chromosome_13 (624.8 ± 58.8); PfW2_chromosome_14 (630.5 ± 98.5); PfW2_apicoplaste (348.2 ± 80.6); and PfW2_mitochondria (7823.6 ± 251.4).

Figure 4

BUSCO completeness results. Pf3D7, PfW2 consensus genomes and the Pf3D7 reference genome were used. The three genomes were annotated with BUSCO and Metaeuk on Galaxy platform [31]. The figure was realized with the BUSCO.py script [32].

Figure 5

Venn diagram showing the number distribution of shared genes between the three Plasmodium falciparum clones. The Venn diagram shows the genes shared by the three strains, whether fragmented or complete. Based exclusively on BUSCO data. The Pf3D7r is the reference genome. Missing genes were not represented. (R script is in Supplementary File S6).

Figure 6

Variability between the Pf3D7 clone sequencing and the Pf3D7 reference genome. (A) Variants observed chromosome by chromosome. The plot was generated with the filtered VCF file (R script is in Supplementary File S7). Each number (1–14) corresponds to a chromosome (1–14) and “A” to the apicoplast. No variant was observed in the mitochondria. (B) Number of SNP substitution for whole genome of Pf3D7 genome reference. (C) Mean quality versus mean depths for the variants observed.