F1000ResearchF1000Research2046-1402F1000 Research LimitedLondon, UK10.12688/f1000research.121402.2Genome NoteArticlesWhole genome sequencing data of
Candida krusei, the pathogen causing Candidaemia, from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia
[version 2; peer review: 2 approved]
FadilahFadilahConceptualizationFormal AnalysisSupervisionWriting – Original Draft Preparationhttps://orcid.org/0000-0002-8120-313812AdawiyahRobiatulConceptualizationFunding AcquisitionResourcesSupervisionWriting – Original Draft Preparationhttps://orcid.org/0000-0002-4987-4839a34WahyuningsihRetnoMethodologyProject AdministrationResourcesSupervision35RozaliyaniAnnaMethodologyResourcesSupervision36FatrilAyu EkaData CurationMethodologyResources3ParamitaRafika IndahData CurationFormal AnalysisMethodologyWriting – Original Draft PreparationWriting – Review & Editinghttps://orcid.org/0000-0002-8166-447912ErlinaLindaData CurationFormal AnalysisMethodologyProject AdministrationWriting – Original Draft PreparationWriting – Review & Editing12IstiadiKhaerunissa AnbarData CurationFormal AnalysisMethodologyProject AdministrationWriting – Original Draft PreparationWriting – Review & Editing2
Department of Medical Chemistry, , Faculty of Medicine, Universitas Indonesia,, Jakarta, Indonesia, 10430, Indonesia
Bioinformatics Core Facilities, Indonesian Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia, 10430, Indonesia
Department of Parasitology, Faculty of Medicine Universitas Indonesia, Jakarta, 10430, Indonesia
Clinical Parasitology Study Programme, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia
Department of Parasitology, School of Medicine, Indonesian Christian University, Jakarta, 13630, Indonesia
Pulmonary Mycosis Centre, Jakarta, 10430, Indonesiabundaadah@gmail.com
Candida krusei is a
Candida non-albicans species with a high prevalence, which causes candidaemia. Current treatment guidelines include fluconazole as a primary therapeutic option for the treatment of these infections; however, it is only a fungistatic against
Candida spp., and both inherent and acquired resistance to fluconazole have been reported.
C.krusei species is also reported as the only
Candida sp. which has an intrinsic resistance factor to fluconazole. Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by
C. krusei. The purpose of this study was to investigate the genome of clinical
C. krusei isolates and correlate the resistant phenotypes with mutations in resistance genes. A total of 16 samples of
C. krusei from clinical samples from hospitals in Jakarta were used in the experiment. All colonies were extracted using the QIAamp DNA Mini Kit. The library was prepared using the Illumina DNA Prep Kit. The sequencing process was carried out on the Illumina MiSeq Platform using a 2x301 paired-end configuration. FASTQ raw files are available under the BioProject Accession Number PRJNA819536 and Sequence Read Archive Accession Numbers SRR18739949 and SRR18739964.
Candida kruseifluconazolegenomic profilingresistance geneUniversitas IndonesiaNKB-1302/UN2.RST/HKP.05.00/2020This study was supported by a PUTI Q1 2020 grant from Universitas Indonesia with grant number: NKB-1302/UN2.RST/HKP.05.00/2020.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Amendments from Version 1
Revisions were made to the Introduction section within the manuscript.
Introduction
Candida krusei (teleomorph
Pichia kudriavzevii) is an opportunistic fungal pathogen.
1 It is the fourth most common non-albicans
Candida species (NAC) causing high prevalence and invasive candidiasis and candidemia.
2,3C. krusei is among unique species with natural resistance towards fluconazole, a commonly used antifungal for
Candida infections.
4 Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by
C. krusei One approach in examining the drug resistance of
C. krusei is by sequencing the genome with potential roles in resistance. Here we report the whole genome sequencing data of
Candida krusei from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia. The sequencing data obtained from this study will help to understand the correlation between phenotypes with mutations in resistance genes and the development of appropriate treatments and medication for the infection.
MethodsSample collection and DNA extraction
The
C. krusei clinical sample were retrieved from the Department of Parasitology Culture Collection, Faculty of Medicine, Universitas Indonesia. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, catalog number: 51304) with bead-beating methods.
5 100 μL culture of
C. krusei were added into 1.5 ml microcentrifuge tube containing 200 μL ATL buffer (Qiagen). Three spoon full of sterilized sand were added into the mixture for bead-beating, which was performed for 10 minutes. 20 μL Proteinase K (Qiagen) were added and the mixture was incubated at 56°C until completely lysed (three hours), and agitated every 10 minutes during incubation time, until homogeneous. 200 μL Buffer AL (Qiagen) were added and the mixture was incubated at 70°C for 10 minutes. 200 μL pure ethanol were added and the mixture was transferred into the QIAamp Mini spin column (Qiagen). Column were centrifugated at 6000 × g for one minute and the flow-through were discarded. QIAamp Mini spin columns were placed innew 2 mL collection tubes and 500 μL Buffer AW1 were added into the column. Columns were centrifugated at 6000 × g for one minute and the flow-through was discarded. QIAamp Mini spin columns were placed into new 2 mL collection tubes and 500 μL Buffer AW2 (Qiagen) were added. Columns were centrifugated at 17000 × g for three minutes and the flow-through was discarded. QIAamp Mini spin columns were transferred into new 1.5 mL microcentrifuge tubes, and 200 μl distilled water were added. Columns were incubated at room temperature for one minute and centrifugated at 6000 × g for one minute to elute the DNA. The quality of extracted DNA (A260/280) was measured using a spectrophotometer (NanoDrop ND-1000). The quantity of extracted DNA was measured with Qubit 4.0 Fluorometer using the dsDNA HS Assay kit.
Library preparation
DNA libraries were prepared using the Illumina DNA Preparation Kit. The index used for the library preparation was Integrated DNA Technologies, Inc (IDT) for Illumina Nextera Indexes for ligation step. The library construction steps were as follows:
Tagmentation
100 ng DNA were added into a 96-well plate and mixed with tagmentation master mix from Illumina Nextera Kit (Illumina). The mix were then incubated at 55°C in a thermal cycler (The Applied Biosystems ProFlex PCR System) for 15 minutes.
Post tagmentation clean-up and amplification of tagmented DNA
Illumina Nextera Kit tagment stop buffer (Illumina) were added to the tagmentation reaction and incubated at 37°C for 15 minutes. The mixture was washed with Illumina Nextera Tagment wash buffer (Illumina) on the magnetic stand. The tagment Wash Buffer was discarded and Nextera PCR master mixes (Illumina) were added onto the beads. Index adapters were added as sample barcoding. The mixture was amplified in thermal cycler.
Libraries clean-up
The beads were added into the supernatant of the mixture and washed twice using 80% ethanol on a magnetic stand. Nextera resuspension buffer (Illumina) reagents were added onto beads and final libraries were retrieved from the supernatant. The libraries of each sample were pooled.
6
Whole genome sequencing
The barcoded DNA libraries were sequenced using an Illumina Miseq Platform on the v3-Flow Cell. The DNA library was denatured according to the manufacturer’s protocol.
7 For quality control, the library was spiked with 1% PhiX Control v3. The sequencing run produced 2 × 301 bp paired-end libraries. The data were deposited to the NCBI Sequence Read Archive (SRA) under BioProject. Total raw reads were obtained using FastQC software, and the total raw bases and percentage of Q30 were evaluated using q30 python scripts.
8
Ethical approval
This research was approved by the Faculty of Medicine Universitas Indonesia Ethical Committee (approval number: 970/UN2.F1/ETIK/PPM.00.02/2021).
Results
The whole raw genome sequence data of 16 clinical isolates of
C. krusei from the Department of Parasitology, Faculty of Medicine of Universitas Indonesia were deposited into NCBI data under BioProject accession number PRJNA819536 and SRA SRR18739949-SRR18739964. The DNA quality and quantity of the samples are shown in
Table 1. Information regarding the raw data is described in
Table 2. These raw data of
C. krusei genome are useful for genome profiling and correlating the resistant phenotypes with mutations in resistance genes.
DNA quantity and quality.
Sample
Concentration (ng/μL)
Purity (A260/A280)
CK1_2021
13.4
2.1
CK2_2021
39.3
2.03
CK3_2021
42.3
2.08
CK4_2021
38.3
2.02
CK5_2021
34.3
1.95
CK6_2021
45.4
1.93
CK7_2021
37
2.07
CK8_2021
23
2.03
CK9_2021
34.3
2.09
CK10_2021
16.7
2.1
CK11_2021
18.9
2.05
CK12_2021
10.3
1.91
CK13_2021
14.2
1.97
CK14_2021
10.1
1.86
CK15_2021
17.9
2
CK16_2021
8.95
1.98
Information of raw sequencing data of
<italic toggle="yes">C. krusei</italic> clinical isolates.
Sample
BioSample accession number
SRA accession number
Q30 (%)
GC content (%)
CK1_2021
SAMN26901813
SRR18739964
89.75
36
CK2_2021
SAMN26901814
SRR18739963
88.98
38
CK3_2021
SAMN26901815
SRR18739956
89.66
38
CK4_2021
SAMN26901816
SRR18739955
90.00
37
CK5_2021
SAMN26901817
SRR18739954
88.83
37
CK6_2021
SAMN26901818
SRR18739953
89.67
37
CK7_2021
SAMN26901819
SRR18739952
89.57
38
CK8_2021
SAMN26901820
SRR18739951
90.40
37
CK9_2021
SAMN26901821
SRR18739950
92.39
37
CK10_2021
SAMN26901822
SRR18739949
89.34
37
CK11_2021
SAMN26901823
SRR18739962
90.65
37
CK12_2021
SAMN26901824
SRR18739961
90.42
37
CK13_2021
SAMN26901825
SRR18739960
93.18
34
CK14_2021
SAMN26901826
SRR18739959
89.01
37
CK15_2021
SAMN26901827
SRR18739958
88.69
37
CK16_2021
SAMN26901828
SRR18739957
89.57
37
Data availabilityUnderlying data
Raw data (FASTQ) files have been deposited into National Center for Biotechnology Information (NCBI,
https://www.ncbi.nlm.nih.gov).
BioProject: Raw sequence reads of
Candida krusei from clinical samples, Accession Number: PRJNA819536;
https://www.ncbi.nlm.nih.gov/bioproject/PRJNA819536
BioSample: Pathogen: clinical or host-associated sample from Pichia kudriavzevii, Accession Number: SAMN26901813;
https://identifiers.org/ncbiprotein:SAMN26901813
BioSample: Pathogen: clinical or host-associated sample from Pichia kudriavzevii, Accession Number: SAMN26901828;
https://identifiers.org/ncbiprotein:SAMN26901828
Raw sequence reads of
Candida krusei from clinical samples:
Sequence Read Archive: Raw sequence reads of Candida krusei from clinical samples, Accession Number: SRR18739949;
https://identifiers.org/insdc.sra:SRR18739949
Sequence Read Archive: Raw sequence reads of Candida krusei from clinical samples, Accession Number: SRR18739964;
https://identifiers.org/insdc.sra:SRR18739964
Acknowledgement
This research is funded by Directorate of Research and Development, Universitas Indonesia under Hibah PUTI 2020 (Grant No. NKB-1302/UN2.RST/HKP.05.00/2020).
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Reference Source10.5256/f1000research.143748.r168114Reviewer response for version 2BanerjeeAtanu1Refereehttps://orcid.org/0000-0001-9319-4593
Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, India
Competing interests: No competing interests were disclosed.
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?
Partly
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?
Yes
Are the rationale for sequencing the genome and the species significance clearly described?
Partly
Are the protocols appropriate and is the work technically sound?
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
10.5256/f1000research.133265.r159413Reviewer response for version 1BanerjeeAtanu1Refereehttps://orcid.org/0000-0001-9319-4593
Amity Institute of Biotechnology,, Amity University Haryana, Manesar, Haryana, India
Competing interests: No competing interests were disclosed.
The article by Fadilah et al, presents the whole genome sequence data of clinical isolates of
C. krusei. While the genome sequence data generated could be useful to advance our knowledge regarding factors that might be responsible for the intrinsic azole resistance exhibited quite often by
C. krusei isolates, there are some significant gaps that need to be filled to make the manuscript more useful for readers. I elaborate on some of them:
The introduction seems extremely brief and does not bring the importance of the work into the proper context. For instance, the authors write "
Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei" and then directly mention "
The purpose of this study was to investigate the genome of clinical isolates of C. krusei and correlate the resistant phenotypes with mutations in resistance genes.". The two statements do not bring the perspective and importance into place appropriately.
Also, there isn't much background literature provided in this section.
The authors mention non-
albicansCandida (NAC) species incorrectly in the abstract and introduction. It should be corrected.
The authors do not provide any background information related to the clinical isolates, except the center. It is extremely important to at least briefly provide some background information on the isolates.
While the authors state that "The purpose of this study was to investigate the genome of clinical isolates of
C. krusei and correlate the resistant phenotypes with mutations in resistance genes", there is no information provided regarding the same. It limits the importance of the paper.
In the keywords, "
Candida krusei" is non-italicized. "Resistance gene" does not make much sense and should be replaced with something more meaningful. The same should also be replaced elsewhere in the MS.
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?
Partly
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?
Yes
Are the rationale for sequencing the genome and the species significance clearly described?
Partly
Are the protocols appropriate and is the work technically sound?
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
10.5256/f1000research.133265.r147383Reviewer response for version 1SunXuepeng1Referee
Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, College of Horticulture Science, Zhejiang A&F University, Hangzhou, China
Competing interests: No competing interests were disclosed.
Drug resistance is a serious problem for human disease. As the human pathogen
C. krusei has natural resistance to the commonly used azole fungicides, it is of particular importance to explore the molecular basis underlying the resistance. This study provides high-depth sequence data for 16 clinic isolates of
C. krusei, which in combination with published
C. krusei genome will provides new information for correlating the mutations with the resistance phenotypes. Due to the potential interest of the data to scientific community and the quality of the manuscript, I would suggest an acceptance of this manuscript in F1000Reseach.
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?
Yes
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?
Yes
Are the rationale for sequencing the genome and the species significance clearly described?
Yes
Are the protocols appropriate and is the work technically sound?
Yes
Reviewer Expertise:
Genomics
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.