DNA was isolated from 100–300 µl of packed animals using the Blood and Tissue DNA isolation kit (QIAGEN, Valencia, CA). The provided protocol was followed with the addition of RNase (4 µl of 100 mg/ml) following the initial lysis for 2 min at room temperature (RT). DNA concentration was determined using the Qubit dsDNA Broad Range Assay Kit (Invitrogen, Carlsbad, CA). Libraries were generated using the Illumina Nextera Sample Prep Kit and indexed using the Nextera Index Kit. A total of 24 uniquely-indexed samples were pooled by mixing 100 ng of each sample. The pooled material was size selected by electrophoresing the DNA on a 2% agarose gel and excising the fragments ranging from 300 to 500 bp. The sample was purified using the QIAGEN MinElute Kit and eluted in 11 µl of Buffer EB. The concentration of the purified sample was determined using the Qubit dsDNA High Sensitivity Assay Kit. Sequencing was performed on the Illumina HiSeq 2500 platform. To increase coverage of some strains, we incorporated data from two separate studies of wild strains (Thompson et al. 2013 (link); Noble et al. 2015 (link)).
Nextera sample prep kit
Manufactured by Illumina
Sourced in United States
The Nextera sample prep kit is a laboratory equipment product from Illumina that enables efficient library preparation for DNA sequencing. It facilitates the generation of sequencing-ready libraries from a variety of input DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Qubit dsdna broad range assay kit, by Thermo Fisher Scientific (1 mentions)
Blood and tissue dna isolation kit, by Qiagen (1 mentions)
Minelute kit, by Qiagen (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Miseq platform, by Illumina (1 mentions)
Clc genomics workbench, by Qiagen (1 mentions)
Miseq pe300 sequencer, by Illumina (1 mentions)
Invisorb spin virus dna mini kit, by Stratec (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nextera index kit, by Illumina (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Bioanalyzer high sensitivity dna chip, by Agilent Technologies (1 mentions)
4 protocols using nextera sample prep kit
1
Illumina Sequencing of Wild Strain DNA
2
Genome Assembly and Annotation of Rhodobacter
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Genome analysis was performed as previously described [15 (link)]. Genomic DNA was isolated from mid-log phase cell culture in marine broth using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA). Samples were prepped for Illumina 250-bp paired-end sequencing using Nextera sample prep kit (Illumina, Inc., San Diego, CA, USA), and sequenced on Illumina MiSeq platform with V2 chemistry. Reads were quality checked and adapter trimmed using Trimmomatic version 0.33 using default parameters for paired-end reads [18 (link)]. Reads were de novo assembled using CLC Genomics Workbench (CLC Bio-Qiagen, Aarhaus, Denmark), and scaffolds generated using MeDuSa [19 (link)] and R. sulfidophilum DSM 2351 as an alignment guide. Alignment of reads to R. sulfidophilum DSM 2351 was performed using Bowtie2 version 2.2.29 [20 (link)] short read mapper, and annotated with National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline [21 (link)].
3
fRuSau02 Phage Genome Sequencing
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fRuSau02 DNA was isolated from crude phage lysate with Invisorb Spin Virus DNA Mini Kit (Stratec Biomedical, Birkenfeld, Germany). Sequencing was performed at the Institute for Molecular Medicine Finland (FIMM) Technology Centre Sequencing Unit [24 ]. For next-generation sequencing, the DNA library was constructed with Nextera sample prep kit (Illumina, San Diego, CA, USA). Paired-end sequencing was done using Illumina MiSeq PE300 sequencer (Illumina, San Diego, CA, USA) with the read length of 300 nucleotides. TheA5 (Andrew And Aaron’s Awesome Assembly)-miseq integrated pipeline for de novo assembly of microbial genomes was used to obtain the genome sequence [25 (link)]. fRuSau02 sequence was submitted to GenBank with accession number MF398190.
4
Bacterial Genomic DNA Extraction and Sequencing
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Genomic DNA of the bacterial isolates was extracted using QIAamp DNA mini kit (Qiagen, Germany) as described by the manufacturer. The quality and quantity of the DNA was checked with Nanodrop 2000c (Thermo Scientific, USA) and Qubit 2.0 fluorometer (Invitrogen, USA), respectively. Sequencing libraries were prepared with Nextera sample prep kit (Illumina, USA) and the indexing was performed according to Nextera Index kit (Illumina, USA). Prior to sequencing, the sequencing library was verified with Bioanalyzer DNA high sensitivity chip (Agilent, USA). Subsequently, 12 pM of the pooled sequencing library was loaded into MiSeq sequencing cartridge (2 × 250 bp, 500 cycles) (Illumina, USA). The sequencing was performed on MiSeq (Illumina, USA) sequencing platform.
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