Whole genome low-coverage short insert size library preparation was performed using the NEBNext Ultra DNA Library Prep Kit (New England Biolabs) and long-range (4422 bp insert size) mate-pair library preparation was done using the Nextera Mate Pair Sample Prep Kit (Illumina Inc.), both according to the manufacturer's protocol. Sequencing was performed on Illumina HiSeq instruments to an average 3x spanning coverage for short insert size paired-end and 38x spanning coverage for mate-pair, with the raw length of the reads displaying a median of 101bp. Structural variants larger than 5,000 bp were detected using the DELLY tool [40 (link)], as previously described [41 (link)]. DNA copy number analysis was performed using qDNAseq [42 (link)]. The sequencing data has been made available through the European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena ; dataset ID: PRJEB9176.)
Nextera mate pair sample prep kit
Manufactured by Illumina
Sourced in United States
The Nextera Mate Pair Sample Prep Kit is a laboratory equipment product designed for the preparation of mate pair libraries for next-generation sequencing. The kit provides a streamlined workflow for generating mate pair libraries from high molecular weight DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (6 mentions)
Biorobot ez1, by Qiagen (5 mentions)
Miseq technology, by Illumina (5 mentions)
Truseq dna pcr free lt sample prep kit, by Illumina (4 mentions)
Nextera xt paired end, by Illumina (4 mentions)
Miseq platform, by Illumina (3 mentions)
Dneasy plant mini kit, by Qiagen (3 mentions)
Truseq dna pcr free sample prep kit, by Illumina (2 mentions)
Ez1 dna tissue kit, by Qiagen (2 mentions)
Miseq sequencer, by Illumina (2 mentions)
Qubit assay, by Thermo Fisher Scientific (2 mentions)
Ez1 dna tissue kit, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Truseq dna pcr free kit, by Illumina (2 mentions)
Miseq reagent kit v2, by Illumina (2 mentions)
Nextseq 500 platform, by Illumina (2 mentions)
Nebnext ultra dna library prep kit, by New England Biolabs (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Smrtbell express template prep kit, by Pacific Biosciences (1 mentions)
Trueseq dna pcr free kit, by Illumina (1 mentions)
25 protocols using nextera mate pair sample prep kit
1
Whole Genome Sequencing Protocol
2
Illumina Sequencing Library Preparation
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Sequencing libraries were prepared from genomic DNA for Illumina MiSeq and HiSeq2500 platforms. A short insert (330 bp) paired-end (PE) library was constructed using a TruSeq DNA PCR-Free LT Sample Prep Kit (Illumina), which reduced PCR amplification bias in library preparation. Mate-paired (MP) libraries with various insert sizes (2, 4, 6, and 8 kbp) were constructed using the Nextera Mate Pair Sample Prep Kit (Illumina). The PE library was sequenced using MiSeq (2 × 230 bp) and the four MP libraries were sequenced using HiSeq2500 (2 × 100 bp).
3
Genome Sequencing and Annotation of Ca. Desulfovibrio trichonymphae
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Sequencing libraries for the ‘Ca. Desulfovibrio trichonymphae' genome were prepared using the TruSeq DNA PCR-free Sample Prep Kit and the Nextera Mate Pair Sample Prep Kit (Illumina, San Diego, CA, USA). Sequencing was performed using the MiSeq Reagent Kit v3 (600 cycles) on an Illumina MiSeq platform. The generated reads were processed for adapter and quality trimming using programs cutadapt and prinseq, respectively (Martin, 2011 ; Schmieder and Edwards, 2011 (link)). The reads were assembled into contigs, using SPAdes 3.0 (Bankevich et al., 2012 (link)), and the contigs and the mate-pair reads were used to generate scaffolds with SCARPA 0.241 (Donmez and Brudno, 2013 (link)). Finding and functional annotation of genes were performed using MiGAP (http://www.migap.org ), and the result was curated manually. Pseudogenes were manually identified as described previously (Hongoh et al., 2008a (link)). Metabolic pathways were reconstructed using the KEGG automatic annotation server (KAAS; Moriya et al., 2007 (link)). Genes were assigned to functional categories based on non-supervised orthologous groups (NOG) (Powell et al., 2014 (link)). Clustered regularly interspaced short palindromic repeat (CRISPR) loci were identified using CRISPRFinder (Grissa et al., 2007 (link)).
4
Genome Sequencing and Comparative Analysis
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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)].
5
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.
6
Genome Sequencing of T. coccineum
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The seeds of T. coccineum (cultivar: Robinson mix) were obtained commercially from Sakata Seed Co., Ltd., Japan (production number, 906435). Genomic DNA was extracted from the seeds using a DNeasy Plant Mini Kit (Qiagen), according to the manufacturer’s instructions. PE and MP libraries with three different insert sizes (3, 5, and 8 kb) of the extracted DNA were constructed using a TruSeq DNA PCR-Free kit (Illumina) and a Nextera Mate-Pair Sample Prep Kit (Illumina), respectively. The PE and MP libraries were then subjected to 151 × 2 cycles of paired-end sequencing, using NovaSeq 6000 Illumina instruments. MS libraries of the extracted DNA were constructed using a TrueSeq DNA PCR-Free kit (Illumina). The MS library was subjected to 301 × 2 cycles of paired-end sequencing using an Illumina Miseq System. PB libraries of the extracted DNA were constructed using a SMRTbell Express Template Prep Kit (PacBio). The PB libraries were subjected to sequencing using a PacBio Sequel II system.
7
Genome Assembly Using Illumina Sequencing
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The Illumina platform (Miseq and Hiseq 2500) was used for sequencing.13 (link) Libraries were prepared according to slight modifications of protocols provided by the manufacturer. Fragmented genomic DNA was further purified using Blue Pippin (Sage Science). A paired-end library consisting of clones ∼720 bp was prepared for the Miseq using a TruSeq DNA PCR-Free LT Sample Prep Kit (Illumina), and 3-kb and 8-kb mate-pair libraries were prepared for the Hiseq 2500 using a Nextera Mate Pair Sample Prep Kit (Illumina), respectively (Supplementary Table S1 ). Longer reads were obtained by using more reagent kits for the Hiseq. K-mer counting and estimation of genome size were performed using JELLYFISH 2.2.0 software.14 (link),15 (link)
Adapter sequences were trimmed from all reads using Trimmomatic-0.30.16 (link) Paired-end reads of high quality (quality value ≥ 20) were assembled de novo using Newbler 2.9 (GS Assembler) to create contigs. Then subsequent scaffolding of the Newbler output was performed using SSPACE 3.0,17 (link) based on Illumina mate-pair information. Gaps inside scaffolds were closed using GapCloser 1.12.18 (link) Diploid sequences of gap-closed scaffolds were merged with Haplomerger-2-20151124.19 (link) CEGMA 2.5 software20 (link) was used to evaluate genome assembly. The mitochondrial genome was generated with the IDBA_UD 1.1.1 assembler.21 (link)
Adapter sequences were trimmed from all reads using Trimmomatic-0.30.16 (link) Paired-end reads of high quality (quality value ≥ 20) were assembled de novo using Newbler 2.9 (GS Assembler) to create contigs. Then subsequent scaffolding of the Newbler output was performed using SSPACE 3.0,17 (link) based on Illumina mate-pair information. Gaps inside scaffolds were closed using GapCloser 1.12.18 (link) Diploid sequences of gap-closed scaffolds were merged with Haplomerger-2-20151124.19 (link) CEGMA 2.5 software20 (link) was used to evaluate genome assembly. The mitochondrial genome was generated with the IDBA_UD 1.1.1 assembler.21 (link)
8
Genomic Characterization of K. pneumoniae Strains
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Genomic DNAs of K. pneumoniae strains were sequenced with the MiSeq Technology (Illumina, San Diego, US) with a 2 × 250 paired-end run strategy using the Nextera Mate Pair sample prep kit (Illumina). Genome assembly was done using the A5-miseq software (http://sourceforge.net/projects/ngopt/ ) and annotated with Prokka (https://github.com/tseemann/prokka ). Plasmid replicons, virulence and resistance genes were sought with Abricate. Genome files were deposited in the National Center for Biotechnology Information (NCBI) database under the Bioproject number PRJNA520974 (Supplementary Table S1 ).
9
Extracting and Sequencing Multi-species Genomes
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DNA was extracted using the CTAB method57 . For each species, two types of libraries, standard shotgun and long-insert (mate pair), were prepared. The shotgun libraries were prepared using the TruSeq DNA sample prep kit and the mate pair library using the Nextera Mate pair sample prep kit (Illumina) following the manufacturer’s instructions for agarose gel based size selection (Supplementary Table 6 ). The libraries were sequenced using HiSeq2000 and Miseq instruments (see Supplementary Table 6 for sequencing parameters and output).
10
Genome Assembly of Testis DNA via Illumina Sequencing
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Genomic DNA was extracted from the testis using a Mag attract HMW DNA kit (Qiagen, Santa Clarita, CA, USA). Paired-end (250 bp) and mate-pair libraries (3, 6, 10, and 15 kb) were constructed using a TruSeq DNA PCR-Free LT Sample Prep Kit and a Nextera Mate Pair Sample Prep Kit (Illumina, San Diego, CA, USA). Sequencing employed an Illumina HiSeq2500. We generated 731,873,830 bp of raw sequence data for de novo genome assembly. Genome assembly into scaffolds was performed using the Platanus v1.2.4 assembler54 after removal of adapter sequences (Platanus_trim and Platanus_internal_trim). Contig assembly was performed using only the PE library, and then scaffolding and gap closure were performed using all libraries. Genome Scope25 (link) was used for genome size and heterozygosity analyses. Generated scaffolds were named in order of length as pvir_s00001, starting with the longest.
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