For genomic DNA, C. albicans cells were grown in YPD at 30 °C and DNA isolated using a Qiagen Genomic Buffer Set, a Qiagen Genomic-tip 100/G or the MasterPure Yeast DNA Purification kit (Epicentre). DNA was sequenced using Illumina HiSeq 2000 generating 101-bp paired reads. Reads were aligned to the SC5314 reference genome using Burrows–Wheeler Aligner (98 ), and converted to sorted BAM format using Samtools (99 (link)). Picard tools (picard.sourceforge.net/ ) were used to preprocess the alignments. The Genome Analysis Toolkit (100 (link)) and Pilon (37 (link)) were used to call variant and reference bases from the alignments. Mutations were inspected in IGV (Broad Institute) and annotated using the VCFannotator (Broad Institute). Ploidy, copy number variation, and LOH analyses were performed as described in SI Appendix, Materials and Methods .
Genomic buffer set
Manufactured by Qiagen
Sourced in Germany
The Genomic Buffer Set is a collection of buffers designed for the purification of genomic DNA. It includes buffers for cell lysis, DNA binding, washing, and elution. The set is intended for use with various DNA extraction and purification methods, but its specific applications and performance characteristics are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (2 mentions)
Genomic tip 100 g, by Qiagen (2 mentions)
Masterpure yeast dna purification kit, by Illumina (1 mentions)
Lyticase, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Sqk lsk109, by Oxford Nanopore (1 mentions)
Exp nbd114, by Oxford Nanopore (1 mentions)
Genomic tips 20 g, by Qiagen (1 mentions)
Exp nbd104, by Oxford Nanopore (1 mentions)
5 protocols using genomic buffer set
1
Genomic DNA Extraction and Sequencing for C. albicans
2
Genomic DNA Extraction and Sequencing
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To extract genomic DNA, ∼109 cells grown overnight in YPD at 30°C were processed using the Qiagen Genomic Buffer Set and the Qiagen Genomic-tip 100/G. Lyticase (Sigma, L2524) was used to enzymatically digest the fungal cell wall. Two libraries were constructed with average insert sizes of 197 bases and 2.5 kilobases (Supplemental Table S2) as previously described (Fisher et al. 2011 (link); Grad et al. 2012 (link)) and were sequenced on an Illumina HiSeq to generate 101 base paired-end reads.
For SNP calling, reads were aligned to the SC5314 genome (version A21-s02-m01-r01;http://www.candidagenome.org ) using BWA 0.5.9 (Li and Durbin 2010 (link)) and variants identified with GATK (McKenna et al. 2010 (link)). Genomic regions exhibiting copy number variation were identified from GC normalized read depth. Potential inversions and translocations identified from alignments of the de novo assemblies were validated by evaluating read support using BreakDancer (Chen et al. 2009 (link)).
The Illumina reads for each strain were assembled using ALLPATHS-LG (Gnerre et al. 2011 (link)), and genes were predicted for each assembly. Both the assemblies and SNPs were used to evaluate variation in gene content and sequence. For detailed DNA sequence analysis methods, see Supplemental Material.
For SNP calling, reads were aligned to the SC5314 genome (version A21-s02-m01-r01;
The Illumina reads for each strain were assembled using ALLPATHS-LG (Gnerre et al. 2011 (link)), and genes were predicted for each assembly. Both the assemblies and SNPs were used to evaluate variation in gene content and sequence. For detailed DNA sequence analysis methods, see Supplemental Material.
3
Fungal Strain Cultivation and DNA Extraction
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A set of 23 black fungal ex-type strains was obtained from CBS-KNAW Fungal Biodiversity Centre and cultivated in Malt Extract Broth (MEB) for 7 d with shaking at 150 rpm at 25 °C (Table S2 ). DNA extraction was performed via a cetyltrimethylammonium bromide (CTAB)-based method and phenol-chloroform/isoamyl alcohol purification (Möller et al. 1992 (link)). Total DNA was purified with Qiagen Genomic Buffer Set and the Qiagen Genomic-tip 100/G. Total RNA was isolated with RNEASY Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The additional strain Cl. carrionii KSF (dH 23894) DNA was obtained from 7-d-old mycelia cultured on Sabouraud Glucose Agar (BBL™) at 25 °C. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen) according to manufacture protocols.
4
High Molecular Weight DNA Extraction and Nanopore Sequencing of Penicillium
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High molecular weight DNA was extracted from the 93 Penicillium isolates using either Genomic Buffer Set (Qiagen, Germany) in combination with QIAGEN Genomic-Tips 20/G or using phenol–chloroform extraction in combination with QIAGEN Genomic-Tips 20/G as described in Petersen et al. (2022 (link)). Specific extraction methods are listed in Additional file 1 : Table S1. Quality control of DNA was performed, followed by a removal of small DNA fragments to increase efficiency of sequencing, and finally another quality control was performed as described in Petersen et al. (2022 (link)). DNA Library preparations of two to four fungi were performed following the Native barcoding genomic DNA (EXP-NBD104, EXPNBD114, and SQK-LSK109) protocol from Oxford Nanopore Technologies (Oxford, UK) and the isolates were sequenced either on a R9.4.1 or R10.3 flow cell (Additional file 1 : Table S1).
5
Whole-genome sequencing of Candida albicans
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To extract genomic DNA, isolates were grown overnight in YPD at 30°C and DNA was isolated from ∼109 cells using a Qiagen genomic buffer set and a Qiagen Genomic-tip 100/G according to manufacturer’s instructions. Libraries were prepared using the Nextera XT DNA library preparation kit protocol (Illumina) with an input of 2 ng/μl in 10 μl. Each isolate was sequenced using Illumina HiSeq 2000, generating 101-bp paired reads. The nuclear genome sequences and General Feature Files (GFF) for C. albicans SC5314 reference genome (version A22) were downloaded from the Candida Genome Database (http://www.candidagenome.org/ ). Alignment, coverage, ploidy, heterozygosity, and variant calling were performed as previously described (84 (link)). Average coverage levels for SC5314, 529L, and CHN1 were 141×, 466×, and 245×, respectively. Heterozygosity plots were constructed using methods from reference 42 (link). Phylogenetic assignment was performed using RAxML (85 (link)) as described in reference 42 (link) and using the isolates from the same study to classify the strains. Large homozygous tracts were confirmed by visual inspection in IGV (86 (link)). Mutations in XOG1 were identified using GATK4 (87 (link)) and manually inspected in IGV. Genetic variants identified between SC5314 and 529L/CHN1 are included in Table S4 in the supplemental material.
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