We present the chromosome sequences of a Naegleria fowleri isolate from a human primary amebic meningoencephalitis (PAM) case. The genome sequences were assembled from Illumina HiSeq and PacBio sequencing data and verified with the optical mapping data.
ABSTRACT
We present the chromosome sequences of a Naegleria fowleri isolate from a human primary amebic meningoencephalitis (PAM) case. The genome sequences were assembled from Illumina HiSeq and PacBio sequencing data and verified with the optical mapping data. This led to the identification of 37 contigs representing 37 chromosomes in N. fowleri.
ANNOUNCEMENT
The free-living ameba Naegleria fowleri (phylum Percoloza; class Heterolobosea) is distributed worldwide in warm freshwater (1, 2). It causes a highly (>97%) fatal brain infection known as primary amebic meningoencephalitis (PAM) (3, 4). The pathogenicity and biology of N. fowleri are poorly understood partly due to a lack of good-quality genome sequences. Two currently available genome sequences of N. fowleri are fragmented and dispersed in 1,124 contigs (5) and 81 contigs (6), respectively. Here, we identified the chromosome sequences of a reference N. fowleri strain (isolate TY).
The N. fowleri isolate TY was from a 1969 primary amebic meningoencephalitis (PAM) patient from Virginia and belongs to genotype III (7). It was maintained under axenic culture conditions. For Illumina HiSeq sequencing, DNA was extracted from approximately 2 × 106 amebas with the DNeasy blood and tissue kit (Qiagen, USA). For library preparation, DNA was sheared using a Covaris LE220 focused ultrasonicator (Covaris, Inc., USA) and cleaned with the AMPure kit (Beckman Coulter, Inc., USA). This was followed by dual-indexing using the NEBNext Ultra library prep reagents (New England Biolabs, USA) and barcoding. Libraries were analyzed for size and concentration, pooled, and denatured for loading onto flow cells for cluster generation. Sequencing was performed on an Illumina HiSeq 2500 instrument using HiSeq rapid SBS v2 250 × 250-cycle paired-end sequencing kits. Illumina reads were filtered using BBDuk (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/) to remove adapters, quality trim to a minimum PHRED score of 20, and remove reads shorter than 50 bp. Additionally, high-molecular-weight (HMW) genomic DNA was purified using the MagAttract HMW DNA kit (Qiagen, USA) and sequenced using the PacBio platform (Pacific Biosciences, USA). DNA libraries were prepared using the SMRTbell template prep kit 1.0 and polymerase binding kit P6, and the filtered reads (minLength = 1,000 bp) were de novo assembled using Canu v1.6. PacBio’s SmrtAnalysis software was used (with default parameters) for adapter trimming and quality control (QC). The resulting consensus sequences were determined with Quiver v1 (https://github.com/PacificBiosciences/GenomicConsensus). The assembly was refined and confirmed by comparison to restriction digest optical maps using the Argus system (OpGen) with MapSolver v2.1.1. The final PacBio assembly of N. fowleri TY was further polished by mapping Illumina reads using the Unicycler polish function (default parameters) in Unicycler v4.4 (8). Default software parameters were used except where otherwise noted.
Mapping the HiSeq Illumina reads (total reads, 5,354,858) to the final PacBio assembly (N50, 10,674; total reads, 396,362) covered 99.97% of the genome with an average coverage depth of 31×. The final assembly of the nuclear genome consisted of 37 contigs comprising 27,994,426 bp, with an N50 value of 756,811 bp and GC content of 36.85%. Each contig contained the characteristic N-terminal and C-terminal telomere sequences, suggesting that these were full-length chromosomes except for one contig for which the C-terminal telomere could not be identified (Table 1). The largest contig/chromosome was 1,206,962 bp, and the shortest was 537,351 bp. A total of 9,405 protein-encoding genes were predicted using BRAKER2 (9). Approximately 75% of the genes contain one or more introns. The average length of an intron is 186 bp. Approximately 5.28% of the total genome contains repetitive sequences as detected using RepeatMasker v4.0.8 and RepeatModeler (default parameters). Discovery of chromosome sequences will help scientists better understand the biology and pathogenicity of this ameba. They will help researchers identify virulence factors in N. fowleri and effective drug targets for treating PAM patients.
TABLE 1.
Chromosome sizes and telomere lengths in the N. fowleri TY isolate
| Chromosome name | Chromosome size (bp) | Telomere length (bp) |
|
|---|---|---|---|
| 5′ telomere | 3′ telomere | ||
| Chr01 | 1,206,962 | 784 | 699 |
| Chr02 | 1,059,554 | 640 | 540 |
| Chr03 | 985,518 | 724 | 615 |
| Chr04 | 984,171 | 322 | 518 |
| Chr05 | 973,969 | 382 | 30 |
| Chr06 | 961,058 | 657 | 840 |
| Chr07 | 853,063 | 69 | 377 |
| Chr08 | 843,297 | 483 | 384 |
| Chr09 | 840,958 | 625 | 710 |
| Chr10 | 833,951 | 723 | 618 |
| Chr11 | 830,534 | 452 | 96 |
| Chr12 | 822,913 | 477 | 456 |
| Chr13 | 800,153 | 349 | 770 |
| Chr14 | 775,976 | 159 | 580 |
| Chr15 | 758,505 | 687 | 400 |
| Chr16 | 756,811 | 646 | 734 |
| Chr17 | 745,360 | 697 | 819 |
| Chr18 | 739,698 | 664 | 281 |
| Chr19 | 725,748 | 770 | 449 |
| Chr20 | 717,112 | 590 | 665 |
| Chr21 | 716,195 | 581 | 599 |
| Chr22 | 713,437 | 727 | 861 |
| Chr23 | 711,928 | 545 | 522 |
| Chr24 | 704,950 | 127 | 530 |
| Chr25 | 667,433 | 702 | 703 |
| Chr26 | 665,971 | 440 | 758 |
| Chr27 | 647,405 | 595 | 682 |
| Chr28 | 638,607 | 538 | 498 |
| Chr29 | 626,360 | 261 | 709 |
| Chr30 | 626,308 | 610 | 0 |
| Chr31 | 614,393 | 16 | 162 |
| Chr32 | 601,738 | 633 | 623 |
| Chr33 | 596,164 | 248 | 394 |
| Chr34 | 592,949 | 689 | 512 |
| Chr35 | 570,195 | 1,022 | 451 |
| Chr36 | 547,731 | 514 | 584 |
| Chr37 | 537,351 | 360 | 383 |
Data availability.
The Naegleria fowleri TY contig/chromosome sequences have been deposited at the National Center for Biotechnology Information’s Sequence Read Archive (SRA) under the accession numbers CP062075, CP062076, CP062077, CP062078, CP062079, CP062080, CP062081, CP062082, CP062083, CP062084, CP062085, CP062086, CP062087, CP062088, CP062089, CP062090, CP062091, CP062092, CP062093, CP062094, CP062095, CP062096, CP062097, CP062098, CP062099, CP062100, CP062101, CP062102, CP062103, CP062104, CP062105, CP062106, CP062107, CP062108, CP062109, CP062110, and CP062111. The raw data are available under the BioSample accession number SRS7483196.
ACKNOWLEDGMENTS
This work was partially supported by an Incubator AMD fund (to Ibne Karim M. Ali), and the efforts of Ibne Karim M. Ali, Sandeep J. Joseph, Shantanu Roy, Jennifer R. Cope, Lori A. Rowe, Mark Burroughs, Mili Sheth, Dhwani Batra, and Vladimir Loparev were funded by the HHS/Centers for Disease Control and Prevention (CDC).
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention.
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Data Availability Statement
The Naegleria fowleri TY contig/chromosome sequences have been deposited at the National Center for Biotechnology Information’s Sequence Read Archive (SRA) under the accession numbers CP062075, CP062076, CP062077, CP062078, CP062079, CP062080, CP062081, CP062082, CP062083, CP062084, CP062085, CP062086, CP062087, CP062088, CP062089, CP062090, CP062091, CP062092, CP062093, CP062094, CP062095, CP062096, CP062097, CP062098, CP062099, CP062100, CP062101, CP062102, CP062103, CP062104, CP062105, CP062106, CP062107, CP062108, CP062109, CP062110, and CP062111. The raw data are available under the BioSample accession number SRS7483196.