Sequence assembly, the subsequent mapping of the raw sequence data, and the analysis of the resulting sequences were done with the Genome Sequencer software suite (v.3.0; Roche) and the Geneious software suite (v.8.1.3; Biomatters Ltd., Auckland, New Zealand). Sequences and their background data were submitted to the European Nucleotide Archive under study accession No. PRJEB19039.
Genome sequencer software suite
Manufactured by Roche
Sourced in New Zealand, Germany
The Genome Sequencer software suite is a comprehensive software package designed for the analysis and interpretation of genomic data. The core function of this product is to facilitate the processing, visualization, and management of DNA sequence information generated from various sequencing platforms. The software provides tools for aligning sequences, identifying genetic variations, and generating reports on the analysis results.
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Lab products found in correlation
Spri te library system, by Beckman Coulter (2 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ampure xp magnetic beads, by Beckman Coulter (2 mentions)
Agencourt rnaclean xp magnetic beads, by Beckman Coulter (1 mentions)
Indices, by Illumina (1 mentions)
Qiaamp minelute virus spin kit, by Qiagen (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
M220 ultrasonicator, by Covaris (1 mentions)
Kingfisher flex system, by Thermo Fisher Scientific (1 mentions)
Ion torrent s5xl instrument, by Thermo Fisher Scientific (1 mentions)
Rnadvance tissue kit, by Beckman Coulter (1 mentions)
Adaptor, by Illumina (1 mentions)
Spriworks fragment library cartridge 2, by Beckman Coulter (1 mentions)
Cdna synthesis system, by Roche (1 mentions)
Rneasy mini spin column, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Newbler, by Roche (1 mentions)
10 protocols using genome sequencer software suite
1
Genome Sequencing and Analysis
2
Viral Genome Enrichment and Sequencing
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qRT-PCR-positive samples were used for high-throughput sequencing (HTS) to obtain more sequence information. A combination of enrichment of viral nucleic acids with RNA baits and following HTS was applied. For this purpose, RNA extracts were subjected to library preparation as detailed described [36 (link)]. Virus nucleic acids were subsequently enriched using myBaits (Arbor Biosciences, Ann Arbor, MI, USA) with the VirBaits panel according to Wylezich et al. [37 (link)] with a hybridization time of 24 hours at 65°C. The bait panel includes 539 baits specific for RVFV. An extended panel (VirBaits 2.0) was used for the dengue virus containing samples that includes 2804 baits specific for dengue virus. The Genome Sequencer software suite (versions 2.6; Roche) was applied to execute reference mapping analyses. Genome sequences of RVFV (KX944814, KX944837, KX944860) and DENV (MT261970, MT981085) were used as reference. Obtained contigs were analyzed with BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) to characterize virus sequences.
3
SARS-CoV-2 Genome Sequencing and Variant Analysis
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Full genome sequences were generated via reference mapping with the Genome Sequencer software suite (version 2.6; Roche; default software settings for quality filtering and mapping), using SARS-CoV-2 strain 2019_nCoV_Muc_IMB1 (accession number LR824570) as reference. Consensus sequences and underlying sequence reads were visualized using Geneious Prime (10.2.3; Biomatters, Auckland, New Zealand). The presence of single nucleotide variants (SNVs) was checked using the variant analysis tool implemented in Geneious Prime (default settings, minimum variant frequency 0.02).
4
RNA Extraction and Sequencing from Cell Culture and Eggs
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RNA from cell culture supernatants or embryonated eggs was extracted using QIAamp MinElute Virus Spin Kit (Qiagen) and further concentration and cleaning was done with Agencourt RNAClean XP magnetic beads (Beckman Coulter). RNA was quantified using Nanodrop UV spectrometer ND-1000 (Peqlab) and used as template for cDNA synthesis with cDNA Synthesis System (REF 11 117 831 001, Roche). Fragmentation of the cDNA applying a target size of 300 bp was done with Covaris M220 ultrasonicator. The sonicated cDNAs were used for library preparation using Illumina indices (Illumina) on a SPRI-TE library system (Beckman Coulter) using SPRIworks Fragment Library Cartridge II (for Roche FLX DNA sequencer; Beckman Coulter) without automatic size selection. Subsequently, upper and lower size exclusion of the library were done with Ampure XP magnetic beads (Beckman Coulter). The libraries were quality checked using High Sensitivity DNA Chips and reagents on a Bioanalyzer 2100 (Agilent Technologies) and quantized via quantitative PCR with Kapa Library Quantification Kit (Kapa Biosystems) on a Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories). Sequencing was done on an Illumina MiSeq using MiSeq reagent kit v3 (Illumina). Raw sequence data were analysed and mapped using the Genome Sequencer software suite (v. 2.8; Roche) and Geneious software suite (v. 6.1.6; Biomatters).
5
SARS-CoV-2 Genome Sequencing and Analysis
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NGS was used to verify the sequence of isolates and isogenic clones prior to experimentation. RNA was extracted using the RNAdvance Tissue kit (Beckman Coulter) and the KingFisher Flex System (Thermo Fisher Scientific). Subsequently, RNA was transcribed into cDNA and sequencing libraries were generated as described27 (link) and were sequenced using the Ion Torrent S5XL Instrument (ThermoFisher). Samples with Ct values >20 for SARS-CoV-2 were additionally treated with RNA baits (myBaits, Arbor Biosciences) for SARS-CoV-2 enrichment before sequencing28 (link). Sequence datasets were analysed by reference mapping with the Genome Sequencer Software Suite (version 2.6, Roche), default software settings for quality filtering and mapping using EPI_ISL_414019 (Alpha), EPI_ISL_2131446 (Alpha) and EPI_ISL_981782 (Beta) as references. To identify potential single nucleotide polymorphisms in the read data, the variant analysis tool integrated in Geneious Prime (2019.2.3) was applied (default settings).
6
Whole Genome Sequencing of Viral RNA
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For whole genome sequencing, viral RNA served as a template for double-stranded cDNA synthesis using the cDNA Synthesis System (Roche, Mannheim, Germany). In summary, RNA was hybridized with random hexanucleotides and subsequently, first strand and second strand syntheses were performed according to the manufacturer’s instructions (Roche, Mannheim, Germany). The Covaris M220 ultrasonicator was used for DNA fragmentation of 0.5-1 µg of double-stranded DNA to an average size of about 300 base-pairs. For library preparation of the fragmented DNA, Illumina adaptors (Biooscientific, Austin, USA) and SPRIworks Fragment Library Cartridge II (Beckman Coulter, USA) were used on a SPRI-TE library system (Beckman Coulter, Fullerton, USA) with manual size selection afterwards. Upper and lower size selection was performed using AMPure XP magnetic beads (Beckmann Coulter). The quality of the library was checked on a Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany) using a High Sensitivity DNA Chip and corresponding reagents. Quantity was determined via qPCR with Kapa Library Quantification Kit (Kapa Biosystems, Cape Town, South Africa). Paired-end sequencing was performed on an Illumina MiSeq using MiSeq reagent kit v3 (Illumina, San Diego, USA). Raw sequence data was analyzed and assembled using the Genome Sequencer software suite (v. 2.8, Roche).
7
Genomic Sequencing of Bank Vole-Derived BvHV Strains
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For the determination of the genomic sequence of original or bank vole passaged BvHV strains (FA07, p1-KS13/920, p1-KS13/914), RNA was extracted from 500 µL BvHV-infected bank vole sera or liver tissue using TRIzol® Reagent (Invitrogen, Darmstadt, Germany) and RNeasy Mini spin columns (Qiagen, Hilden, Germany) as previously described [36 (link)]. cDNA synthesis and library preparation were performed as previously described [37 (link)]. Sequencing was performed with an Illumina MiSeq instrument (Illumina, San Diego, CA, USA). Raw sequence data were analyzed and mapped using the Genome Sequencer software suite (v. 2.8; Roche, Mannheim, Germany) and Geneious software suite (v. 6.1.6; Biomatters, Auckland, New Zealand). Classification of the BvHV isolates was performed by phylogenetic analyses of complete genome sequences including reference strains MN242371.1 and KC411777.1 [25 (link)] using maximum-likelihood methods in MEGA5 [38 (link)] (Table S6 ).
8
De Novo Sequence Assembly
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For sequence assembly, reads representing the respective sequences were selected by mapping the complete data set to reference sequences using the Genome Sequencer software suite (v2.6; Roche). Subsequently, the sorted reads were used for a de novo assembly using newbler (Roche) and the complete raw data sets were mapped along the resulting consensus sequences in order to identify potential sequencing errors. Thereafter, the sequences were visually inspected using Geneious (v6.1.7; Biomatters, Auckland, New Zealand).
9
Metagenomics Analysis Using RIEMS and 18S rRNA
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Generated metagenomics datasets were analysed using a combination of the RIEMS tool (Scheuch et al. 2015 (link)) and reference mapping against 18S rRNA gene sequences of suspected candidates using the Genome Sequencer software suite (versions 2.6; Roche) as described by Wylezich et al. (2019 ). Accession numbers of the sequences used as references are included in the Supplementary Table S1 . Different identity thresholds (95–99%) and a minimum overlap length of reads of 95% were applied for reference mapping. The obtained 18S rRNA sequences were verified by Blast analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome ). Whereas for the proof-of-concept study (Wylezich et al. 2019 ) only nearly complete 18S rRNA sequences extracted from metagenomics datasets were rated as positive, in the present study, no minimum fragment length of the obtained contigs was presupposed for detection but instead each single read was considered positive if it was a specific hit.
The 18S rRNA sequences generated within this study were submitted to GenBank when the fragments were longer than 1000 base pairs (seeSupplementary Table S1 ). They are accessible under the accession numbers MN914072-MN914086.
The 18S rRNA sequences generated within this study were submitted to GenBank when the fragments were longer than 1000 base pairs (see
10
Viral Genome Variant Analysis
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Variant analyses were performed to compare within-sample variant frequencies in the RABV genome of sample L03007 and the KBLV genome of sample L02374 before and after VirBaits treatment. The whole virus genomes of RABV and KBLV were received by mapping the sequencing datasets against the lyssavirus reference sequences (NC_001542.1), and by de-novo assembly and subsequent mapping, respectively, using the Genome Sequencer software suite (version 3.0; Roche). Subsequently, the obtained whole genome sequence was used in a second mapping round as reference sequence in order to calculate the total number of viral reads for the respective datasets. To identify potential single nucleotide variants (SNPs), the Torrent Suite plugin Torrent variantCaller (version 5.12) was used (parameter settings: generic, S5/S5XL(530/540), somatic, low stringency, changed alignment arguments for the TMAP module from map 4 [default] to map1 map2) while the previously determined whole genome sequence was set as reference. In addition, the variant analysis tool integrated in Geneious Prime (2019.2.3) was applied for further confirmation and the acquisition of variant frequencies for each variant (default settings, minimum variant frequency 0.02). Positions of identified potential SNPs were visually inspected in Geneious Prime.
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