An aliquot of yeast strain W303 was obtained from Dr. Gholson Lyon (CSHL). Four-milliliter cultures in 15-mL Falcon tubes of yeast were grown in YPD overnight at 32°C to ∼1 × 108 cells. The cells were purified using the Gentra Puregene Yeast/Bacteria kit (Qiagen). DNA was stored at −20°C for no more than 7 d prior to use.
Gentra puregene yeast bacteria kit
Manufactured by Qiagen
Sourced in Germany, Australia
The Gentra Puregene Yeast/Bacteria kit is a laboratory equipment product designed for the purification of genomic DNA from a variety of yeast and bacterial samples. It provides a rapid and efficient method for extracting high-quality DNA suitable for downstream applications such as PCR, sequencing, and genotyping.
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Lab products found in correlation
Onestep pcr inhibitor removal kit, by Qiagen (2 mentions)
Nd 100, by Qiagen (2 mentions)
Jumpstart taq, by Merck Group (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Qiaprep spin and midi kits, by Qiagen (2 mentions)
Redtaq, by Thomas Scientific (2 mentions)
Cloneamp hifi, by Takara Bio (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Miniseq system, by Illumina (1 mentions)
Miniseq, by Illumina (1 mentions)
Genomic tip 20 g, by Qiagen (1 mentions)
Sqk rbk004, by Oxford Nanopore (1 mentions)
Nextera xt dna library prep kit, by Illumina (1 mentions)
Dneasy powerlyzer microbial kit, by Qiagen (1 mentions)
Miseq control software, by Illumina (1 mentions)
Miseq personal sequencer, by Illumina (1 mentions)
Crimson taq dna polymerase, by New England Biolabs (1 mentions)
Wizard genomic purification kit, by Promega (1 mentions)
27 protocols using gentra puregene yeast bacteria kit
1
Yeast Strain W303 DNA Extraction
2
Genome Sequencing and Mutation Analysis of E. coli
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DNA was isolated from overnight cultures using the Gentra Puregene Yeast/Bacteria kit (Qiagen). Twenty-one multiplex libraries were made using the IntegenX Apollo 324™ System. The libraries were sequenced on a single lane of an Illumina HiSeq, to a read length of 140 bases, resulting in an average fold-coverage of 400 for each strain. The reads were mapped to the E. coli K-12 MG1655 reference genome (GenBank: U00096.2) [22 (link)] using BWA for Illumina [23 (link)]. The resulting SAM files were converted to BAM files using SAMtools [24 (link)]. The BAM files were visualized using Integrated Genome Viewer (IGV) [25 (link)], manually searching the genomes for SNPs, deletions, insertions, and amplifications. An insertion is predicted at a genomic location with a marked decrease in read depth, which occurs when individual sequencing reads fail to span that location. An amplification is predicted at genomic intervals containing a consistent read depth of more than 1.5-times the background read depth. All mutations are described in Additional file 1 : Table S1. To reduce the possibility of overlooking some genomic mutations, the sequence data were also analyzed with FreeBayes, a powerful variant-detector package [26 ]. No additional mutations were identified using this second method.
3
Hybrid Assembly of Bacterial Genome Using Illumina and Nanopore
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Hybrid assembly was performed using short-read Illumina MiniSeq (Illumina) and long-read Nanopore MinION (Oxford Nanopore Technologies). WGS of strain KUHS13 was performed using a MiniSeq system (Illumina) with a High Output reagent kit (300 cycles). The library for sequencing (insertion size, 500 to 900 bp) was prepared using a Nextera XT DNA library prep kit (Illumina). On the other hand, the DNA library for Nanopore MinION was prepared using a rapid barcoding kit (SQK RBK-004; Oxford Nanopore Technologies) from total DNAs extracted using a Qiagen Genomic-tip 20/G or Gentra Puregene yeast/bacteria kit (Qiagen, Hilden, Germany) and then sequenced on a MinION flow cell (R9.4.1). WGS statistics are shown in Table S1 in the supplemental material. Raw data sets from the Nanopore MinION assay were submitted to Porechop (v0.2.3). The reads were assembled de novo using Canu (v1.8) (30 (link)). After the data were trimmed from Canu, they were polished with Racon (v1.3.1.1) and Pilon (v1.20.1) (31 (link), 32 (link)). The nucleotide sequence of the left end of the linear plasmid (pELF2) was further checked by Sanger sequencing. DFAST and RAST were used to obtain the annotation (33 (link), 34 (link)).
4
Draft Genome Sequencing of Aeromonas veronii
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Sequencing and assembly of draft genomes of 25 A. veronii strains were conducted as described in our previous study [28 (link)]. Briefly, bacterial DNA was extracted using Gentra Puregene Yeast/Bacteria Kit (Qiagen, Chadstone, Victoria, Australia). Briefly, the DNA libraries were sequenced via the 150 bp or 250 bp paired-end sequencing chemistry on the MiSeq Personal Sequencer [29 (link)]. Reads were assembled using Shovill (v 1.0.5), and genome coverage was calculated using qualimap (v 2.2.1) [30 (link)]. Sequencing of the draft genome was performed in the Marshall Centre for Infectious Diseases Research at the University of Western Australia.
5
Bacterial Growth Adductomics Protocol
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The exposure was done similar to that in the bacterial growth assay. Experiments were done in triplicates. For S. oneidensis, 21.9 mL bacterial suspension was used per replicate and 135 mL was used for B. subtilis. Bacterial growth was monitored before and after exposure. After 8 or 5 hours of exposure for S. oneidensis and B. subtilis, respectively, cell pellets were collected by centrifugation at 4000 × g for 20 minutes and stored at −80 °C until being thawed for DNA extraction. DNA extraction of S. oneidensis was done using Gentra Puregene Yeast/Bacteria Kit (Qiagen) and that of B. subtilis was extracted using a DNeasy PowerLyzer Microbial Kit (Qiagen). Both protocols were optimized to be suitable for the adductomics study. See ESI† for details of cell lysis and DNA extraction.
6
Sequencing of Campylobacter concisus Genomes
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C. concisus strains sequenced in this study were isolated from saliva samples or intestinal biopsies in our previous studies3 (link)6 (link)11 (link)22 (link). The genomes of 27 C. concisus strains were sequenced. C. concisus strains were grown on Horse Blood Agar (HBA) plates as previously described1 (link). DNA was extracted from each C. concisus strain using the Gentra Puregene Yeast/Bacteria Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The quality of DNA was checked using Nanodrop and Qubit Fluorometer. Bacterial genomic DNA (1 ng) was used for genomic library generation in accordance with the Nextera XT protocol (Ver. May 2012). Libraries were sequenced for a 250 bp paired-end sequencing run using Nextera XT V2 on the MiSeq Personal Sequencer running version 1.1.1 MiSeq Control Software (Illumina Inc., San Diego, CA, USA). Reagent contamination was controlled by barcoding all DNA samples and preparation of barcoding index primers for a single use. The quality of reads was assessed based on the Phred quality score of the reads. The reads mapping fold coverage was calculated using qualimap_v2.040 (link). We aimed to get a fold coverage of at least 50X for each genome, which was shown to be adequate for characterization of genomes41 (link).
7
Bacterial Diversity in Deoxynivalenol Degradation
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To analyze the bacterial population diversity and its change at high concentrations of DON, the enriched microbial consortium C20 was isolated from environmental samples and was added to MM to degrade DON, with DON concentrations of 10 and 100 μg ml−1. The bacteria were incubated in media with different concentrations of DON at 30°C for 72 h. Genomic DNA of the enriched microbial cultures was extracted and purified with Gentra Puregene Yeast/Bacteria Kit (Qiagen Inc., Mississauga, Ontario, Canada). The hypervariable V3-V4 region of the 16S rRNA gene was amplified by PCR with primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC) (Ahad et al., 2017 (link)) and sequenced by Sangon Biotech Co. (Shanghai, China). Further, the 16S rRNA (V3-V4 region) gene sequences were compared with the ribosomal database project (RDP)1 . All treatments were conducted in triplicates.
8
Bacterial DNA Extraction and 16S rRNA Sequencing
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Genomic DNA from each culture was extracted and purified using the Gentra Puregene Yeast/Bacteria Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocols for Gram-positive and/or Gram-negative bacteria when applicable. Genomic DNA was amplified using modified bacterial specific 16S ribosomal RNA (rRNA) gene primers 8F (5′-GTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′) [61 (link),62 ] and directly sequenced at the University of Chicago’s Comprehensive Cancer Center’s DNA Sequencing and Genotyping Facility using their methods. We assembled the resulting forward and reverse sequence reads and manually edited these in Geneious Prime build 29 November 2019 [63 (link)]. The resulting consensus sequences were subsequently queried against the nr database at NCBI (National Center for Biotechnology Information), using a BLASTn search [64 (link)] to retrieve the sequence and GenBank numbers of the most closely related taxa for phylogenetic placement of NTK sequences.
9
Sequence Analysis of mecA and ftsZ Genes
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Genomic DNA (gDNA) was extracted from 1 ml of overnight cultures using the Gentra Puregene Yeast/Bacteria kit (Qiagen). PCR was carried out on gDNA to determine the sequence of the mecA PNA target region (i.e. SD region and 5′ end of mecA) with primers mecA-99F and mecA-528R for both NCTC 13142 and HH-1 (Additional file 1 : Table S1). To determine the sequence of the ftsZ PNA target regions (i.e. SD region and 5′ end of ftsZ), primers E15ftsZ_F and E15ftsZ_R specific for NCTC 13142, and ED99ftsZ_F and EDftsZ_R specific for HH-1 were used (Additional file 1 : Table S1). PCR was carried out with Crimson Taq DNA polymerase (New England Biolabs) with cycling conditions of 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 52 °C (mecA primers) or 61 °C (ftsZ primers) for 30 s, 68 °C for 1 min, and a final extension of 68 °C for 5 min.
10
Genomic DNA Extraction from Neisseria gonorrhoeae
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Total genomic DNA was extracted from N. gonorrhoeae isolates using the Gentra Puregene Yeast/Bacteria Kit, following the manufacturer’s instructions for DNA purification from Gram-negative bacteria (Qiagen, Valencia, CA, USA). DNA concentration and purity were determined with a NanoDrop ND-100 spectrophotometer (Bio-Active Co., Ltd, Bangkok, Thailand), and DNA samples were stored at -20°C until use.
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