Genomic DNA was extracted using the EZ1 biorobot and the EZ1 DNA tissue kit (Qiagen) and then sequenced on a MiSeq sequencer (Illumina, San Diego, France) with the Nextera Mate Pair sample prep kit (Illumina) and Nextera XT Paired End (Illumina), as previously described [46 (link)]. The assembly was performed using Spades V. 3.15 [47 (link)] and trimmed (using Trimmomatic v. 0.36) [48 (link)]. Scaffolds of < 800 bp and scaffolds with a depth value < 25% of the mean depth were removed. The best assembly was selected using criteria such as the number of scaffolds, N50, and number of N. All assembled genomes were annotated using Prokka v 1.14.5 (Fontainebleau, France) [49 (link)]. The gff3 output files were used to construct a core genome alignment using Roary v3.13.0 (USA) using default parameters [50 (link)]. Comparative analysis was performed for the five placental isolates and reference strains such as RSA493 (Genbank: NC_002971.4), Z3055 9Genbank: NZ_LK937696), NL3262 (Genbank: NZ_CP013667), and Guiana (Genbank: HG825990.30). The phylogenetic tree was reconstructed by using FastTree (Berkeley, CA, USA) [51 (link)].
Nextera xt paired end
Manufactured by Illumina
Sourced in United States
The Nextera XT Paired End is a high-throughput library preparation solution for Illumina sequencing platforms. It utilizes a tagmentation-based approach to simultaneously fragment and tag DNA samples, enabling efficient library construction for paired-end sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Nextera mate pair sample prep kit, by Illumina (4 mentions)
Biorobot ez1, by Qiagen (4 mentions)
Ez1 dna tissue kit, by Qiagen (3 mentions)
Miseq technology, by Illumina (3 mentions)
Ez1 dna tissue kit, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Miseq sequencer, by Illumina (1 mentions)
Qubit assay, by Thermo Fisher Scientific (1 mentions)
Nextera mate pair, by Illumina (1 mentions)
Miseq instrument, by Illumina (1 mentions)
5 protocols using nextera xt paired end
1
Genome Sequencing and Comparative Analysis
2
Hybrid Genome Sequencing Approach
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Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, Les Ulis, France) with the EZ1 DNA tissue kit and then sequenced on the MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT Paired end (Illumina, San Diego, CA, USA), as previously described [23 (link)]. In order to improve the genome sequence, an Oxford Nanopore approach was performed on 1D genomic DNA sequencing for the MinIon device using an SQK-LSK109 kit. The library was constructed from 1 µg genomic DNA without fragmentation and end repair. Adapters were ligated to both ends of genomic DNA. After purification on AMPure XP beads (Beckman Coulter Inc, Fullerton, CA, USA), the library was quantified by a Qubit assay with the high sensitivity kit (Life technologies, Carlsbad, CA, USA). A total of 1047 active pores were detected for the sequencing and the WIMP workflow was chosen for bioinformatic analysis in real time. After 1 h of run time and end life of the flowcell, 617,960 reads were generated as raw data.
3
Hybrid Genome Sequencing Workflow
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Genomic DNA was extracted with the EZ1 biorobot (Qiagen, Courtaboeuf, Les Ulis, France) with the EZ1 DNA tissue kit and then sequenced on the MiSeq technology (Illumina, San Diego, CA, USA) using the the Nextera Mate Pair sample prep kit and Nextera XT Paired end (Illumina, San Diego, CA, USA), as previously described [23 (link)]. In order to improve the genome sequence, the Oxford Nanopore approach was operated on 1D genomic DNA sequencing for the MinIon device using SQK-LSK109 kit. A library was then constructed from 1 µg genomic DNA without fragmentation and end repair. Adapters were ligated to both ends of genomic DNA. After purification on AMPure XP beads (Beckman Coulter Inc, Fullerton, CA, USA), a Qubit assay quantified the library with the high sensitivity kit (Life technologies, Carlsbad, CA, USA). In total, 1159 active pores were detected for sequencing, and the workflow WIMP was chosen for live bioinformatic analysis. After 1.5 h run time and end-of-life of the flowcell, 82,460 reads as raw data were generated.
4
Genome Assembly and Comparison Protocol
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The total DNA of the genomes was recovered using the EZ1 biorobot (Qiagen, Courtaboeuf, France) and the EZ1 DNA tissue kit. Sequencing was performed using MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit and Nextera XT paired end (Illumina), as previously described (Morel et al. 2015 (link)). Several bioinformatic tools, including Velvet (Zerbino and Birney 2008 (link)), Spades (Bankevich et al. 2012 (link)), and SOAPdenovo (Luo et al. 2012 (link)) were used to assemble the genomes. GapCloser software (Xu et al. 2019 (link)) was used to reduce assembly gaps. Scaffolds which had fewer than 800 base pairs (bp) or had a depth value lower than 25% of the mean depth were removed. The best assembly was selected using different criteria (number of scaffolds, N50, number of N). The degree of genomic similarity of each strain was evaluated by processing sequences using the Orthologous ANI Tool (OAT) software (Lee et al. 2016 (link)). Furthermore, the Genome-to-Genome Distance Calculator (GGDC) web server, which is available online (http://ggdc.dsmz.de ), was used to calculate digital DNA–DNA hybridisation (dDDH) values between the genomes of closest species, as previously described (Meier-Kolthoff et al. 2013 (link)).
5
Genomic DNA Extraction and Comparative Analysis
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Genomic DNA extraction was realized using the EZ1 DNA tissue kit (Qiagen, Hilden, Germany) adapted to EZ1 biorobot. It was sequenced by the MiSeq instrument (Illumina Inc, San Diego, CA, USA) using the Nextera Mate Pair and Nextera XT Paired End (Illumina) sample preparation kit, following the same protocol previously used 19 . Three known softwares were used to correctly assemble this genome, including Spades 20 , Velvet 21 and Soap Denovo 22 . To manage trimmed or untrimmed sequences, MiSeq and Trimmomatic softwares were used, respectively 23 . In addition, we used the GGDC (Genome-to-Genome Distance Calculator) web server available online (http://ggdc.dsmz.de) to calculate the genomic similarities 24 . This allowed us to obtain DNA-DNA hybridization (DDH) values of these compared genomes. The average nucleotide identity (OrthoANI) was also accessed using the OAT software 25 .
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