Genomic DNA (gDNA) was obtained from 7-day-old liquid culture of P. insidiosum (strain Pi-S) using our previously described protocol [21]. Harvested hyphae (1,000 mg wet weight) were ground in a mortar after adding liquid nitrogen. Resulting hyphal power was moved to a sterile 50-mL tube containing 5 ml of the extraction solution [250 mM NaCl, 100 mM Tris–HCl (pH 8.0), 100 mM ethylenediaminetetraacetic acid (pH 8.0), and 1% sodium dodecyl sulfate] and RNase A (final concentration: 50 µg/ml). The cell lysate was incubated (with gentle inversion) at 37 °C for 2 hr before adding proteinase K (final concentration: 50 mg/mL). After the sample was incubated at 56 °C overnight, the supernatant was collected by centrifugation (10,000 xg) at room temperature for 30 min, mixed with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) solution, gently inverted for 15 min, and centrifuged (2,000 xg) at 25 °C (room temperature) for 30 min. The resulting aqueous phase was moved to a new 50-ml tube, mixed with the same volume of isopropanol, gently inverted 10 times, and centrifuged (2,000 xg) at 25 °C for 5 min. A resulting gDNA pellet was collected, washed with 70% ethanol, air dried, and resuspended in 5 mM Tris-HCl (pH 8.0). Extracted gDNA was kept at -20 °C and transported the following day to the National Omics Center, National Science and Technology Development Agency, Pathum Thani, Thailand, for genome sequencing.
Genome sequencing was done following the method of Shearman et al. [22]. In brief, a P. insidiosum gDNA sample was purified using the Ampure PB beads (Pacific Biosciences, Menlo Park, USA) and checked for DNA integrity using the Pippin Pulse Electrophoresis System (Sage Science, Beverly, USA). The gDNA sample (10 ng) was torn using a Covaris gTube (4,500 rpm for 2 min) for library preparation (using a ‘0.75%DF Marker S1 high-pass 15–20 kb’ BluePippin cassette with 12-50kb selection) according to the Pacific Biosciences protocol (20 kb Template Preparation Using BluePippin Size-Selection). Genome sequencing was conducted on the PacBio RSII. Raw reads with a length of at least 20 kb were defined as seed reads, which were corrected by shorter reads (sizes < 20 kb) using the Pacific Biosciences SMRT analysis software v2.3.0 (the RS_PreAssembler.1 protocol with default settings).
PacBio-derived corrected reads (2,632.0 Mb) were assembled using CANU (v2.1) [23], a software that shows an ability to produce highly contiguous assemblies. Three rounds of polishing were carried out using ARROW [24] (SMRT link version 5.0.1). The first initial assemblies were aligned with the original raw PacBio reads using pbmm2 (v1.2.1) (https://github.com/PacificBiosciences/pbmm2). The resulting alignment was then used for calling the consensus sequence using ARROW. Gene prediction was made with the Funannotate pipeline (v1.7.4) (https://github.com/nextgenusfs/funannotate). As a result, a total of 840 contigs (maximum length: ~1.3 Mb; minimum length: 1,294 bases; N50: 229.9 Kb; L50: 70) were obtained. The sequence assembly showed a total genome length of 66.7 Mb (178x coverage) containing 57.2% G-C content and 20,375 ORFs. The genome completion estimated using BUSCO (v4.0.2) [25] and the Fungi Ogb10 dataset (containing 250 conserve eukaryotic genes) was 85.5%. All contig sequences have been deposited in the NCBI database under the accession numbers BBXB02000001 - BBXB02000840 via the project PRJDB3817 (Table 1).
Table 1: Overview of data files/data sets.
Label
|
Name of data file/data set
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File types
(file extension)
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Data repository and identifier (DOI or accession number)
|
Data file 1
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P. insidiosum strain Pi-S, whole genome shotgun sequencing project
|
FASTA
|
BBXB02000001 - BBXB02000840
|