Draft Genome Sequence of Aduncisulcus paluster, a Free-Living Microaerophilic Eukaryote Belonging to the Fornicata

概要

GENOME SEQUENCES

Draft Genome Sequence of Aduncisulcus paluster, a Free-Living
Microaerophilic Eukaryote Belonging to the Fornicata
Ikuko Yuyama,a

Keitaro Kume,b Takumi Tamura,c Yuji Inagaki,c,d Tetsuo Hashimotoc,e

Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, Japan

a

Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

b
c

Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan
Center for Computational Sciences, University of Tsukuba, Ibaraki, Japan

d

Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan

e

Ikuko Yuyama and Keitaro Kume contributed equally to this work. Author order was determined in order of seniority.

ABSTRACT Aduncisulcus paluster is a free-living, unicellular flagellate belonging to the
eukaryotic lineage Fornicata, which includes free-living and commensal/parasitic organisms.
Here, we report the draft genome sequence of A. paluster, which provides clues for
elucidating the adaptation to microaerophilic/anaerobic environments and the transition
between free-living and commensal/parasitic lifestyles in Fornicata.

he Fornicata include various microaerophiles/anaerobes, which possess mitochondrionrelated organelles. Aduncisulcus paluster (1) is a free-living fornicate that shares a common
ancestor with the parasites (2). Previously, only transcriptome data were available for A. paluster,
which contain many sequences derived from bacteria in the culture medium (2). Therefore,
to obtain more accurate genetic information, we performed genome/transcriptome analyses
on A. paluster cells purified by density gradient centrifugation (3). This is the third genomic
analysis of free-living fornicates (3, 4).
We have maintained the laboratory culture of A. paluster NY0171, which is identical to
the strain NIES-1843 (1). The culture containing its food bacteria was kept at 17.5°C in a
modified TYGM-9 medium (10%) prepared with filtered seawater. We inoculated from a single A. paluster culture to fresh medium in three flasks (1,650 mL in total) and cultured the
cells for 1 week. The cells in two out of the three flasks were combined and used for RNA
extraction, and the cells in the other flask were applied to DNA extraction. The cells were
collected by density gradient centrifugation using the 0, 10, and 20% Optiprep (Sigma) gradient at 17°C, 800  g, for 20 min (3). The purified cells at 3.4  107 and 1.56  106 were
used for RNA and DNA extractions, respectively. DNA was extracted using the SDS plus phenol-chloroform-isoamyl alcohol (25:24:1) method (5), while RNA was extracted using TRIzol
reagent (Sigma) according to the manufacturer’s instructions. We constructed the libraries
for transcriptome sequencing (RNA-seq) and genome sequencing (Genome-seq) analyses
using the Kapa stranded mRNA-seq kit for RNA and the Kapa HyperPrep kit (Kapa
Biosystem) for DNA, respectively. Prior to the library construction, the DNA sample was
sheared into 500 bp by an ultrasonicator (Covaris). Then, Genome-seq and RNA-seq analyses (150-bp paired-end) were performed on HiSeqX and NextSeq500 (Illumina) at a biotech
company (SeibutsuGiken Inc.).
We obtained 463,267,674 reads/69.9 Gbp (Genome-seq) and 223,709,682 reads/31.8
Gbp (RNA-seq). We used the default parameters for subsequent analyses unless otherwise
specified. Reads were preprocessed using Fastp v0.21.0 with a .Q30 threshold (6),
followed by de novo assembly performed in MaSuRCA v3.3.3 (Genome-seq) (7) and
Trinity v2.12 (RNA-seq) (8). For gene prediction, we executed Braker2 v2.1.6 (9–15) with
the supporting information generated by aligning the cleaned RNA-seq reads to the
February 2023 Volume 12 Issue 2

Editor Jason E. Stajich, University of California,
Riverside
Copyright © 2023 Yuyama et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Keitaro Kume,
keitaro_kume@md.tsukuba.ac.jp.
The authors declare no conflict of interest.
Received 3 June 2022
Accepted 5 January 2023
Published 25 January 2023

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TABLE 1 Overview of nuclear genome sequences for fornicates
Description
Aduncisulcus paluster
Carpediemonas membranifera
Kipferlia bialata
Spironucleus salmonicida
Giardia intestinalis

Genome
size (Mb)
29.4
24.2
51.0
12.9
12.8

No. of
contigs
25,863
69
11,563
233
211

N50
(kbp)
4.6
905.8
10.5
150.8
2,762.4

GC
(%)
39.1
57.1
49.4
33.5
49.2

No. of predicted
proteins
15,316
8,300
17,389
8,067
5,901

Mean amino
acid (aa) length
418.5
467.2
333.0
373.0
530.0

No. of
introns
11,743
124,912
3
8

Reference(s)
This study
4; this study
3
3, 4
3, 4

MaSuRCA assembly by TopHat v2.1.1 (16) (option: -g 1), and we used TransDecoder v5.5.0
(https://github.com/TransDecoder/TransDecoder) for the Trinity assembly. Based on the
BLASTN/BLASTP (17, 18) searches using the assembled/predicted sequences against the
NCBI nt/nr databases (ver. Dec-21-2021/Feb-04-2022), we removed the putative contaminated sequences that matched to the top hit database sequences from outside the phylum, Metamonada, that includes the Fornicata, with an E value of ,1e210 and pident
(percentage of identical matches) of .0.95. Then, we assessed genome completeness of
the predicted sequences by BUSCO v5.1.2 with the eukaryote ODB10 data set (19), and it
was 40.4% (complete, 30.2%; fragment, 10.2%). We finally summarized the statistics in
Table 1. Both predicted protein sequences were annotated by InterProScan v5.52 (20, 21)
and by BLASTP search against nr (if pident was .0.9 and qcov (query coverage) was .0.9,
the best-hit annotation was used).
This draft genome will help in the study of the genome evolution associated with the evolutionary transition between free-living and commensal/parasitic lifestyles in the Fornicata.
Data availability. The genome/transcriptome sequences are available at DDBJ/
ENA/GenBank (sra-run DRR351251/DRR353576) under accession no. BQXS01000001
to BQXS01023235/ICSK01000001 to ICSK01036959.

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ACKNOWLEDGMENTS
Computations were partially performed on the NIG supercomputer at ROIS National
Institute of Genetics.
This work was supported in part by grants from the Japan Society for the Promotion
of Science (19KK0185 and 22K06368 awarded to T.H.) and by the “Tree of Life” research
project of the University of Tsukuba.

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