Transcriptome data sets of free-living diplomonads, Trepomonas sp. and Hexamita sp.

ABSTRACT

Most species belonging to the diplomonad genera, Trepomonas and Hexamita, are considered to have secondarily adapted to free-living lifestyles from the parasitic ancestor. Here, we report the annotated transcriptome data of Trepomonas sp. NIES-1444 and Hexamita sp. NIES-1440, the analysis of which will provide insights into the lifestyle transitions.

ANNOUNCEMENT

Diplomonadida, a microaerophilic/anaerobic protist subgroup of Fornicata, comprises various parasitic/commensal genera. Interestingly, most species of the genera Trepomonas and Hexamita are considered to have secondarily adapted from parasitic to free-living lifestyles (1, 2). Since the sequence data of these species are crucial for understanding lifestyle transitions, we conducted RNA-seq analyses on Trepomonas sp. and Hexamita sp. and annotated these assemblies. We also annotated the existing transcriptome assembly of a free-living fornicate, Kipferlia bialata (3).

We obtained Trepomonas sp. NIES-1444 and Hexamita sp. NIES-1440 strains from the NIES collection. The cultures were maintained at 17°C in UYTS + rice medium (by ultrapure water; https://mcc.nies.go.jp/02medium-e.html#uyts_rice), and each protist was mass-cultured in flasks with fresh medium (500 mL) with its diverse food bacteria maintained in the culture medium for 1 week.

For RNA extraction, cells were collected by the following methods.

For Hexamita sp., density-gradient centrifugation was performed at 17°C, 800 × g for 20 min using the 0%, 10%, 20%, and 70% Optiprep (Sigma) gradient modified from the previous method (3). Purified cells were processed for RNA extraction with TRIzol and Turbo DNA-free Kit (Ambion), followed by PolyA selection using Dynabeads mRNA Purification Kit (Ambion). Following SMART-Seq v.4 Ultra Low Input RNA Kit for Sequencing (Illumina), we constructed libraries for RNA-seq analysis with Nextera XT DNA Library Prep Kit (Illumina) and analyzed these on a MiSeq (Illumina, 300-bp paired end).

For Trepomonas sp., cells were collected by two-round centrifugation at 4°C, first with 2,030 × g for 20 min, then 4,670 × g for 5 min. Using TRIzol, we extracted total RNA and provided 100 µg for Eurofins Genomics [polyA-selected RNA-seq, HiSeq2000 (Illumina), TruSeq SBS Kit v.3, 100-bp paired end].

We obtained 50,863,282 and 291,258,186 reads for Hexamita sp. and Trepomonas sp. Default parameters were used except where otherwise noted (see doi: 10.5281/zenodo.8246431). Reads were preprocessed using Trimmomatic v.0.39 [option: ILLUMINACLIP: (TruSeq3-PE-2.fa/NexteraPE-PE.fa) :3:30:10; LEADINGL: 30; TRAILING: 30; SLIDINGWINDOW: 4:25; MINLEN: 36] (4) and assembled using Trinity v.2.15.0 (option: max_memory: 200 G, CPU: 128) (5). ORFs (open reading frames) were determined for both assemblies using TransDecoder v.5.5.0 (https://github.com/TransDecoder/TransDecoder) (option: genetic_code: Hexamita, m: 50). We removed putative contaminants based on BLASTP (6, 7) against NCBI-nr v20221219 if they met all of the following criteria for the BLASTP top hit: e value of ≤1e-10, percentage of identical matches of ≥0.95, and query coverage per subject of ≥0.5, except when Metamonada NCBI: txid2611341 was hit in the hit list with an e value of ≤1e-10.

To compare the transcriptomes of these free-living diplomonads with another free-living fornicate, we also analyzed the Trinity assembly of K. bialata reported in the previous study (3). These three resultant assemblies were annotated by the best-hit annotation of InterProscan v.5.52 (8, 9) or BLASTP against the local database (Uniprot v.20230125 + Trichomonas/Giardia/Trepomonas/Spironucleus data: PRJNA16084/1439/288252/60811). If both BLASTP and InterProScan annotations were available, we used the former if its e value was ≤1e-40; otherwise, we chose the latter. Non-annotated CDSs (coding sequences) of ≥300 aa were added as hypothetical proteins. Final statistics, including assembly completeness (protein-mode BUSCO v.5.1.2) (10), are in Table 1.

TABLE 1.

Overview of transcriptome sequences for the three fornicates

Organism		Kipferlia bialata	Trepomonas sp. NIES-1444	Hexamita sp. NIES-1440
Predominant bacterial genera (top 3) contaminated in culture (percentage of sequences per genus against all bacterial sequences)		Pseudoalteromonas (100)b	Undibacterium (38.9), Chryseobacterium (20.0), Sulfurospirillum (12.5)	Cutibacterium (85.5), Ralstonia (2.3), Alcanivorax (1.1)
No. of bases sequenced (Gb)		27.6b	29.1	7.6
No. of assembled transcripts		20,294b	76,247	48,794
No. of bases in assembled transcripts (kb)		28,897b	61,267	38,374
No. of annotated CDS regions		10,283	37,247	24,666
GC content of CDS regions (%)		56.9	43.1	45.9
N50 of CDS regions (bp)		2,820	1,466	1,295
Average length of annotated proteins (aa)		516.3	370.1	328.9
BUSCO completeness for annotated proteins (data set: eukaryote odb 10)	Completea	33.4 (S:31.8, D:1.6)	20.4 (S:11.4, D:9.0)	15.3 (S:10.6, D:4.7)
	Fragment	12.2	7.5	9.0
	Missing	54.4	72.1	75.7
Reference	Accession no.	PRJDB5457 and DRR083618	PRJDB15716 and DRR460931	PRJDB15717 and DRR460932
	Publicationb	Reference (3)	–	–

^a D, duplicate; S, single.

^b Numbers updated from original report; –, none, this study.

DATA AVAILABILITY

BioProject numbers are PRJDB15716/PRJDB15717 (Trepomonas/Hexamita). Raw sequence and TSA (Transcriptome Shotgun Assembly) data are available at DDBJ/ENA/GenBank under accession numbers DRR460931/DRR460932 (Trepomonas/Hexamita) and ICTH01000001-ICTH01037247/ICTI01000001-ICTI01024666/ICTJ01000001-ICTJ01010283 (Trepomonas/Hexamita/Kipferlia).