LGAAP: Leishmaniinae Genome Assembly and Annotation Pipeline.

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

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 GenBank

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

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

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).