DNA was extracted using High Pure Viral Nucleic Acid Large Volume Kit (Roche Diagnostics; Cat. 05 114 403 001), with some modifications. Briefly, bone's fragments were ground, and the powder was resuspended in 1 mL of binding buffer supplemented with poly(A) carrier RNA and proteinase K. Samples were incubated overnight at 37°C. The subsequent steps were performed according to the manufacturer's instructions.
High pure viral nucleic acid large volume kit
Manufactured by Roche
Sourced in Switzerland, Germany
The High Pure Viral Nucleic Acid Large Volume Kit is a laboratory product designed for the isolation and purification of viral nucleic acids from large volume samples. It provides a reliable and efficient method for extracting viral RNA or DNA from various biological sources.
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Lab products found in correlation
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (4 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Quantitect whole transcriptome kit, by Qiagen (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Qiagen (1 mentions)
Miseq platform, by Illumina (1 mentions)
Amicon ultra 15 30 kda, by Merck Group (1 mentions)
Nextera dna cd indexes, by Illumina (1 mentions)
Nextera dna flex library preparation, by Illumina (1 mentions)
Nextseq high output kit v2, by Illumina (1 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Access rt pcr system, by Promega (1 mentions)
Rnase 1, by Thermo Fisher Scientific (1 mentions)
Miseq reagent kit v2 500 cycle, by Illumina (1 mentions)
Miseq next generation sequencing platform, by Illumina (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Midori green dna stain, by Nippon Genetics (1 mentions)
Milli q water, by Merck Group (1 mentions)
22 protocols using high pure viral nucleic acid large volume kit
1
Viral DNA Extraction from Bone Fragments
2
Ancient DNA Extraction from Lung Nodule
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Sampling of the lung nodule, extraction, and library preparation were conducted in dedicated ancient DNA clean rooms at the Max Planck Institute for the Science of Human History in Jena, Germany. The nodule was broken using a hammer, and a 5.5 mg portion of the nodule was taken with lung tissue for extraction according to a previously described protocol with modifications [18 (link)]. The sample was first decalcified overnight at room temperature in 1 mL of 0.5 M EDTA. The sample was then spun down, and the EDTA supernatant was removed and frozen. The partially decalcified nodule was then immersed in 1 mL of a digestion buffer with final concentrations of 0.45 M EDTA and 0.25 mg/mL Proteinase K (Qiagen) and rotated at 37 °C overnight. After incubation, the sample was centrifuged. The supernatants from the digestion and initial decalcification step were purified using a 5-M guanidine-hydrochloride binding buffer with a High Pure Viral Nucleic Acid Large Volume kit (Roche). The extract was eluted in 100 μl of a 10-mM tris-hydrochloride, 1-mM EDTA (pH 8.0), and 0.05% Tween-20 buffer (TET). Two negative controls and one positive control sample of cave bear bone powder were processed alongside LUND1 to control for reagent/laboratory contamination and process efficiency, respectively.
3
Comprehensive Sequencing and Annotation of Pseudomonas Phages
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Phage DNA isolation was carried out using the High Pure Viral Nucleic Acid Large Volume Kit (Roche Cat No. 05114403001). We sequenced the phage genomes on Illumina MiSeq platform and achieved coverages of at least 50x. The sequences were filtered by BBduk1 and assembled using Spades (v.3.13.1) with default settings and the “careful” option (Nurk et al., 2013 (link)). From the output, we used the “contigs.fasta” files for annotation with Prokka (v.1.14.6) (Seemann, 2014 (link)). We downloaded all other Pseudomonas phage gene sequences from Uniprot and used them as trusted sequences for annotation. Phage phylogeny was carried out using VICTOR (Meier-Kolthoff and Göker, 2017 (link)), and comparison was performed by Brig (Alikhan et al., 2011 (link)). The phage sequences were uploaded to the NCBI database (Bioproject: PRJNA720536 ).
4
Optimized DNA Extraction from Bone Powder
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DNA extraction in both laboratories was carried out using a silica-based method optimized for the recovery of highly degraded DNA63 (link),64 (link). To release DNA from 50–100 mg of bone powder, a solution of 900 μl EDTA, 75 μl H2O and 25 μl proteinase K was added. In a rotator, samples were digested for at least 16 h at 37 °C, followed by an additional hour at 56 °C. The suspension was then centrifuged and transferred into a binding buffer. To bind DNA, large-volume silica spin columns (High Pure Viral Nucleic Acid Large Volume Kit; Roche Molecular Systems) were used. After two washing steps using the manufacturer’s wash buffer, DNA was eluted in TET (10 mM Tris, 1 mM EDTA and 0.05% Tween 20). At the Max Planck Institute for the Science of Human History, the second elution of DNA from the spin column was carried out using a fresh aliquot of elution buffer for a total of 100 µl DNA extract, whereas at the Max Planck Institute for Evolutionary Anthropology the same aliquot of elution buffer was loaded twice for a total of 50 µl DNA extract (protocol: 10.17504/protocols.io.baksicwe).
5
Ancient DNA Extraction from Dental Pulp
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All laboratory procedures were performed in the dedicated ancient DNA facilities of the Max Planck Institute for the Science of Human History in Jena, Germany.
Teeth from nine individuals (one tooth from each), buried in the Mikhaylovka II tombs of the Samara region in Russia, were sectioned in the cementoenamel junction using a coping saw and 50–100 mg of dental pulp was removed from each tooth using a dental drill.
Extraction of 50–60 mg of dental pulp from each tooth sample was performed using a previously described protocol; optimised for the recovery of short DNA fragments, most typical of ancient DNA53 (link). An initial lysis step was performed over a 12–16 h incubation of the dental pulp powder in 1 ml of extraction buffer (0.45 M EDTA, pH 8.0, and 0.25 mg ml−1 proteinase K) at 37 °C. Following extraction, DNA was bound to a silica membrane using a binding buffer containing guanidine hydrochloride (protocol previously described in ref. 53 (link)) and purified in combination with the High Pure Viral Nucleic Acid Large Volume Kit (Roche). DNA was eluted in 100 μl of TET (10 mM Tris-HCl, 1 mM EDTA, pH 8.0 and 0.05% Tween20). One extraction blank and one positive extraction control (previously assessed cave-bearing specimen) were taken along for the extraction slot.
Teeth from nine individuals (one tooth from each), buried in the Mikhaylovka II tombs of the Samara region in Russia, were sectioned in the cementoenamel junction using a coping saw and 50–100 mg of dental pulp was removed from each tooth using a dental drill.
Extraction of 50–60 mg of dental pulp from each tooth sample was performed using a previously described protocol; optimised for the recovery of short DNA fragments, most typical of ancient DNA53 (link). An initial lysis step was performed over a 12–16 h incubation of the dental pulp powder in 1 ml of extraction buffer (0.45 M EDTA, pH 8.0, and 0.25 mg ml−1 proteinase K) at 37 °C. Following extraction, DNA was bound to a silica membrane using a binding buffer containing guanidine hydrochloride (protocol previously described in ref. 53 (link)) and purified in combination with the High Pure Viral Nucleic Acid Large Volume Kit (Roche). DNA was eluted in 100 μl of TET (10 mM Tris-HCl, 1 mM EDTA, pH 8.0 and 0.05% Tween20). One extraction blank and one positive extraction control (previously assessed cave-bearing specimen) were taken along for the extraction slot.
6
Extracting Ancient DNA from Tooth Roots
7
SARS-CoV-2 Viral Sequencing from Plasma
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Six hundred μL of patient plasma were pre-treated with Turbo DNAse (Turbo DNA-free kit, ThermoFisher Scientific, Brazil) aiming host/bacterial DNA removal. After DNAse inactivation, 5 to 6 individual samples were mixed in a single pool. The total pool quantity was extracted using the High Pure Viral Nucleic Acid Large Volume kit (Roche, Brazil) following the manufacturer's instructions. The extracted samples were submitted to reverse transcription using the Superscript III First-Strand Synthesis System (ThermoFisher Scientific). The amplification was performed using the QuantiTect Whole Transcriptome kit (QIAGEN, Brazil). Genomic libraries were prepared using the Nextera DNA Flex Library Preparation and Nextera DNA CD Indexes kits (Illumina, USA). The sequencing was performed in Illumina NextSeq 500 sequencer using the NextSeq High Output Kit v 2.5, 300 cycles (Illumina).
8
Extraction of Ancient DNA from Bone Powder
9
Comprehensive Sewage-based Norovirus Detection
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Total RNA was extracted from 1 milliliter of the concentrated sewage
using High Pure Viral Nucleic Acid Large Volume Kit (Roche) according to the
manufacturer’s procedure. Reverse transcription-PCR (RT-PCR) was
performed using Access RT-PCR System (Promega, USA). Primer pair G1-SKF (CTG CCC
GAA TTY GTA AAT GA) and G1-SKR (CCA ACC CAR CCA TTR TAC A) was used for
amplification of a 330-nucleotide (nt) GI sequence corresponding to nt position
5342 to 5671 of strain Norwalk/68/US (accession no., M87661), and primer pair
COG2F (CAR GAR BCN ATG TTY AGR TGG ATG AG) and G2-SKR (CCR CCN GCA TRH CCR TTR
TAC AT) were used for amplification of a 387-nt GII sequence corresponding to nt
position 5003 to 5389 of strain Lordsdale/93/UK (accession no., X86557). The
amplified sequences encompass the 3′ end of ORF1 to the beginning of
the capsid region37 (link). In order to detect cross contamination, a
RT-PCR reaction using the RNA extracted from 3% beef extract solution served as
a blank control, and a negative control containing all the components of the
reaction except for the template was also included.
using High Pure Viral Nucleic Acid Large Volume Kit (Roche) according to the
manufacturer’s procedure. Reverse transcription-PCR (RT-PCR) was
performed using Access RT-PCR System (Promega, USA). Primer pair G1-SKF (CTG CCC
GAA TTY GTA AAT GA) and G1-SKR (CCA ACC CAR CCA TTR TAC A) was used for
amplification of a 330-nucleotide (nt) GI sequence corresponding to nt position
5342 to 5671 of strain Norwalk/68/US (accession no., M87661), and primer pair
COG2F (CAR GAR BCN ATG TTY AGR TGG ATG AG) and G2-SKR (CCR CCN GCA TRH CCR TTR
TAC AT) were used for amplification of a 387-nt GII sequence corresponding to nt
position 5003 to 5389 of strain Lordsdale/93/UK (accession no., X86557). The
amplified sequences encompass the 3′ end of ORF1 to the beginning of
the capsid region37 (link). In order to detect cross contamination, a
RT-PCR reaction using the RNA extracted from 3% beef extract solution served as
a blank control, and a negative control containing all the components of the
reaction except for the template was also included.
10
Phage DNA Extraction and Sequencing Protocol
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High-titer phage suspensions (1012–1014 PFU/ml) were used for DNA extraction. Prior to the extraction, bacterial lysates containing phages were treated with DNase I (80 U/ml; Thermo Scientific, USA) and RNase I (80 μg/ml; Thermo Scientific, USA) at 37 °C for 3 h to remove non-phage nucleic acids. Phage DNA was then extracted using a High Pure Viral Nucleic Acid Large Volume Kit (Roche, Mannheim, Germany) with initial phage capsid disruption by treatment with proteinase K and 0.5% sodium dodecyl sulfate (Sigma-Aldrich) for 2 h at 56 °C. The integrity of the extracted DNA was determined by electrophoresis in 0.7% agarose stained with Midori Green DNA Stain (Nippon Genetics Europe, Germany). The concentration of DNA was determined with a Biowave II UV/Vis spectrophotometer (Biochrom WPA, UK) and purity was determined in terms of the ratio 260/280 nm. Phage genomes were sequenced with the Illumina MiSeq next-generation sequencing platform (Genomed SA. Poland) using MiSeq Reagent Kit v2 500-cycles (Illumina, USA). Sequencing quality was assessed on the basis of average base quality, GC content and adapter contamination [31 ]. All sequenced phages were assembled into one unique contig and sequence assembly was conducted with the Shovill pipeline and assembly improvement pipeline [32 ]. Genome assemblies were annotated with Prokka [33 ].
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