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. 2021 Jul 22;10(29):e00439-21. doi: 10.1128/MRA.00439-21

LGAAP: Leishmaniinae Genome Assembly and Annotation Pipeline

Hatim Almutairi a,b, Michael D Urbaniak a, Michelle D Bates a, Narissara Jariyapan c, Godwin Kwakye-Nuako d, Vanete Thomaz-Soccol e, Waleed S Al-Salem b, Rod J Dillon a, Paul A Bates a, Derek Gatherer a,✉
Editor: Irene L G Newtonf
PMCID: PMC8297458  PMID: 34292068

ABSTRACT

We present the LGAAP computational pipeline, which was successfully used to assemble six genomes of the parasite subfamily Leishmaniinae to chromosome-scale completeness from a combination of long- and short-read sequencing data. LGAAP is open source, and we suggest that it may easily be ported for assembly of any genome of comparable size (∼35 Mb).

ANNOUNCEMENT

We developed an automated genome assembly and annotation pipeline, successfully applying it to six genomes in the parasite subfamily Leishmaniinae, namely, (i) Leishmania martiniquensis (MHOM/TH/2012/LSCM1, LV760), (ii) Leishmania orientalis (MHOM/TH/2014/LSCM4, LV768), (iii) Leishmania enriettii (MCAV/BR/2001/CUR178, LV763), (iv) Leishmania sp. Ghana (MHOM/GH/2012/GH5, LV757), (v) Leishmania sp. Namibia (MPRO/NA/1975/252, LV425), and (vi) Porcisia hertigi (MCOE/PA/1965/C119, LV43). This paper closes the “protocol gap” (1) for this project by making all methods fully available.

The pipeline was written and executed using the Snakemake (2) workflow management system and consists of a total of 314 computational steps, divided into 21 sequential processes in two main phases (Fig. 1). Genomic DNA was extracted from a previously developed culture system for L. orientalis axenic amastigotes (3) and sequenced using two standard technologies, i.e., short read (Illumina) and long read (Oxford Nanopore Technologies [ONT]).

FIG 1.

FIG 1

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Graphical representation of the LGAAP protocol.

The first (assembly) phase of the pipeline comprises eight sequential processes, i.e., (i) long-read assembly using Flye (version 2.8.2) (4), (ii) mapping of short reads onto assemblies using Minimap2 (version 2.17) (5), (iii) creation of consensus sequences using SAMtools (version 1.11) (6), (iv) polishing of assemblies using Pilon (version 1.23) (7), (v) revision of consensus sequences using SAMtools, (vi) ordering and orientation of the chromosomes and breakage of any chimeric sequences using RaGOO (version 1.1) (8), (vii) sorting and removal of any duplicated scaffolds or contigs using Funannotate (version 1.5.3) (9), and (viii) generation of a quality report using QUAST (version 5.0.2) (10).

The second (annotation) phase of the pipeline comprises 13 sequential processes, i.e., (i) scanning of assemblies for vector contamination using BLAST+ (version 2.10.1) (11) against UniVec (12), (ii) masking of contaminants using BEDTools (version 2.30) (13), (iii) quality statistics preannotation using AGAT (version 0.6.0) (14), (iv) detection of repeats using RepeatModeler (15) running from Dfam TE Tools Container (version 1.3.1) (16), (v) classification of transposable elements using TEclass (16) running from a docker container (version 2.1.3b) (17), (vi) masking of identified complex repeats using RepeatMasker (version 4.1.2-p1) (18), (vii) downloading of protein and transcript evidence from TriTrypDB (release 47) (19), (viii) evidence-based annotation using MAKER2 (20) running from a docker container (version 2.31.10) (21), (ix) quality checking of annotation using GenomeTools (version 1.2.1) (22) and GAAS (version 1.2.0) (23), (x) ab initio annotation using AUGUSTUS (version 3.3.2) (24) within MAKER2, (xi) repeating of the ninth step, (xii) annotation assignments using BLAST+ against UniProt (25) and InterProScan (version 5.22-61.0) (26), and (xiii) finalization of the longest isoforms of each predicted protein using AGAT.

The final product of the analysis pipeline is five files per genome, i.e., the chromosome-scale assembly, proteins, and transcripts in FASTA format and two general feature format (GFF) files, one containing the coordinates of each feature and one with the longest isoforms. Testing on genomes longer than 35 Mb is a future optimization priority. Comparison of the performance of LGAAP with all 50 Leishmania genome assemblies in GenBank is shown in Table 1.

TABLE 1.

Assembly metrics for Leishmania genome assemblies deposited in GenBanka

Organism NCBI assembly no. Strain Sequencing technology(ies) Assembly method No. of scaffolds Total length (bp) N50 (bp)
L. aethiopica GCA_003992445 209-622 PacBio RS II CANU 118 33,648,436 763,733
L. aethiopica GCA_000444285 L147 Illumina Allpaths-LG 160 31,630,816 1,001,864
L. amazonensis GCA_003992505 210-660 PacBio RS II CANU 92 33,504,997 850,106
L. amazonensis GCA_000438535 NA Roche 454, Illumina Newbler, Velvet, Zorro 2,627 29,029,348 22,901
L. amazonensis GCA_005317125 UA301 Illumina SMALT 34 32,156,470 NA
L. arabica GCA_000410695 LEM1108 Illumina AllPaths-LG 168 31,269,090 1,057,807
L. braziliensis GCA_003304975 IOC-L 3564 IonTorrent SPAdes 1,029 38,003,648 758,103
L. braziliensis GCA_000340355 MHOM/BR/75/M2903 Roche 454 Newbler 744 35,210,150 1,030,512
L. braziliensis GCA_000002845 MHOM/BR/75/M2904 Sanger NA 138 32,068,771 992,961
L. braziliensis GCA_900537975 MHOM/BR/75/M2904 PacBio, Illumina NA 35 32,301,632 NA
L. chagasi GCA_014466975 MCER/BR/1981/M6445/Salvaterra Illumina SOAPdenovo 36 31,924,566 1,043,794
L. chagasi GCA_014466935 MHOM/HD/2017/M32502/Amapala Illumina SOAPdenovo 36 31,924,975 1,043,719
L. donovani GCA_000470725 BHU 1220 Illumina Bowtie 36 32,414,853 1,024,085
L. donovani GCA_000227135 BPK282A1 Roche 454, Illumina NA 36 32,444,968 1,024,085
L. donovani GCA_003730175 FDAARGOS_360 PacBio, Illumina CANU 71 34,011,430 828,097
L. donovani GCA_003730215 FDAARGOS_361 PacBio, Illumina CANU 56 33,453,722 1,033,854
L. donovani GCA_900635355 HU3 Illumina NA 36 33,035,865 NA
L. donovani GCA_000283395 Ld 2001 SOLiDb Velvet 14,518 27,466,456 3,370
L. donovani GCA_000316305 Ld 39 SOLiD Velvet 16,323 23,683,296 1,772
L. donovani GCA_003719575 LdCL PacBio, Illumina HGAP, Celera Assembler, CANU 36 32,959,864 NA
L. donovani GCA_001989955 MHOM/IN/1983/AG83 Illumina AllPaths, STLab-assembler 36 32,148,377 1,015,993
L. donovani GCA_001989975 MHOM/IN/1983/AG83 Illumina AllPaths 36 32,196,393 1,029,368
L. donovani GCA_002243465 Pasteur PacBio HGAP 37 33,545,875 1,079,609
L. enriettii GCA_000410755 LEM3045 Illumina AllPaths-LG 495 30,761,861 868,233
L. enriettii* GCA_017916305* MCAV/BR/2001/CUR178, LV763 ONT, Illumina LGAAP 54 33,318,864 1,075,649
L. gerbilli GCA_000443025 LEM452 Illumina AllPaths-LG 492 31,398,648 379,527
L. guyanensis GCA_003664525 204-365 PacBio RS II CANU 123 33,816,023 683,170
L. infantum GCA_003671315 HUUFS14 Illumina ABySS 2,507 32,578,914 29,848
L. infantum GCA_000002875 JPCM5 Sanger NA 76 32,122,061 1,043,848
L. infantum GCA_900500625 JPCM5 PacBio, Illumina NA 36 32,803,248 NA
L. infantum GCA_003020905 TR01 Illumina Geneious 36 32,009,138 NA
L. lainsoni GCA_003664395 216-34 PacBio RS II CANU 137 34,152,029 638,860
L. major GCA_000002725 Friedlin Sanger NA 36 32,855,089 NA
L. major GCA_000331345 LV39c5 Roche 454 Newbler 849 32,327,517 978,401
L. major GCA_000250755 SD 75.1 Roche 454 Newbler 36 31,242,750 1,022,795
L. martiniquensis GCA_000409445 LEM2494 Illumina AllPaths-LG 251 30,813,970 873,628
L. martiniquensis* GCA_017916325* MHOM/TH/2012/LSCM1, LV760 ONT, Illumina LGAAP 42 32,413,670 1,046,741
L. mexicana GCA_003992435 215-49 PacBio RS II CANU 55 32,057,209 825,953
L. mexicana GCA_000234665 MHOM/GT/2001/U1103 Sanger NA 588 32,108,741 1,044,075
L. orientalis* GCA_017916335* MHOM/TH/2014/LSCM4, LV768 ONT, Illumina LGAAP 98 34,194,276 1,120,138
L. panamensis GCA_000340495 MHOM/COL/81/L13 Illumina SOAP denovo 952 31,263,945 156,905
L. panamensis GCA_000755165 MHOM/PA/94/PSC-1 Roche 454, Illumina Newbler, PAGIT 35 30,688,794 1,043,456
L. peruviana GCA_001403695 LEM-1537 NA NA 37 33,890,200 1,047,715
L. peruviana GCA_001403675 PAB-4377 NA NA 37 32,907,781 1,015,393
Leishmania sp. GCA_000981925 AIIMS/LM/SS/PKDL/LD-974 Illumina A5 assembly pipeline 1,100 27,848,322 61,709
Leishmania sp. Ghana* GCA_017918215* MHOM/GH/2012/GH5, LV757 ONT, Illumina LGAAP 116 35,953,538 1,100,365
Leishmania sp. Namibia* GCA_017918225* MPRO/NA/1975/252, LV425 ONT, Illumina LGAAP 67 34,118,624 1,066,046
L. tarentolae GCA_009731335 Parrot Tar II PacBio RS II HGAP 179 35,416,496 663,019
L. tarentolae GCA_009770625 Parrot Tar II Roche 454 Newbler 7,227 31,556,583 7,432
L. tropica GCA_011316065 ATCC 50129 Illumina CLC Genomics Workbench 1,928 30,870,161 32,161
L. tropica GCA_014139745 CDC216-162 PacBio RS II, Illumina Flye 43 32,700,668 1,070,514
L. tropica GCA_000410715 L590 Illumina AllPaths-LG 448 32,989,014 303,214
L. tropica GCA_003067545 MHOM/LB /2017/IK Illumina CLC NGS Cell 9,499 32,139,927 13,854
L. tropica GCA_003352575 MHOM/LB/2015/IK Illumina CLC NGS Cell 17,013 32,280,712 7,721
L. turanica GCA_000441995 LEM423 Illumina AllPaths-LG 336 32,320,007 397,299
Porcisia hertigi* GCA_017918235* MCOE/PA/1965/C119, LV43 ONT, Illumina LGAAP 74 34,958,538 967,170
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a

Asterisks indicate the six genomes assembled using LGAAP. NA, either not applicable to the technology used or not available from the GenBank record.

b

SOLiD, sequencing by oligonucleotide ligation and detection.

Data availability.

Genomes assembled using this protocol are available in the NCBI Assembly database with the following accession numbers: L. martiniquensis, GCA_017916325.1; L. orientalis, GCA_017916335.1; L. enriettii, GCA_017916305.1; Leishmania sp. Ghana, GCA_017918215.1; Leishmania sp. Namibia, GCA_017918225.1; and Porcisia hertigi, GCA_017918235.1. Raw sequencing data are available with the following NCBI BioProject accession numbers: L. martiniquensis, PRJNA691531; L. orientalis, PRJNA691532; L. enriettii, PRJNA691534; Leishmania sp. Ghana, PRJNA691536; Leishmania sp. Namibia, PRJNA689706; and Porcisia hertigi, PRJNA691541. The workflow is available at GitHub (https://github.com/hatimalmutairi/LGAAP) and Zenodo (https://doi.org/10.5281/zenodo.4663265).

ACKNOWLEDGMENT

This work is funded by a Ph.D. studentship grant to H.A. from the Saudi Arabian Ministry of Health.

Contributor Information

Derek Gatherer, Email: d.gatherer@lancaster.ac.uk.

Irene L. G. Newton, Indiana University, Bloomington

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Genomes assembled using this protocol are available in the NCBI Assembly database with the following accession numbers: L. martiniquensis, GCA_017916325.1; L. orientalis, GCA_017916335.1; L. enriettii, GCA_017916305.1; Leishmania sp. Ghana, GCA_017918215.1; Leishmania sp. Namibia, GCA_017918225.1; and Porcisia hertigi, GCA_017918235.1. Raw sequencing data are available with the following NCBI BioProject accession numbers: L. martiniquensis, PRJNA691531; L. orientalis, PRJNA691532; L. enriettii, PRJNA691534; Leishmania sp. Ghana, PRJNA691536; Leishmania sp. Namibia, PRJNA689706; and Porcisia hertigi, PRJNA691541. The workflow is available at GitHub (https://github.com/hatimalmutairi/LGAAP) and Zenodo (https://doi.org/10.5281/zenodo.4663265).

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