Tumor DNA and RNA were extracted independently from each fresh frozen clinical specimen using the DNeasy Blood and Tissue kit (Qiagen, CA, #69504) or RNeasy Plus Mini kit (Qiagen, CA, #74134), respectively, following established protocols. Total RNA and DNA yields and concentrations were determined by Qbit. Indexed libraries for MPseq (1µg DNA) and RNAseq (100ng total RNA) were generated using the Nextera Mate-Pair Kit (Illumina, CA, FC-132–1001) or the TruSeq RNA Access Library Prep Kit (Illumina, CA, RS-301–2001), as previously described, following the manufacturer’s instructions16 (link). Libraries were sequenced on the Illumina HiSeq4000 platform at a depth of four or seven libraries per lane, respectively.
Nextera mate pair kit
Manufactured by Illumina
Sourced in United States
The Nextera Mate Pair Kit is a library preparation kit designed for sequencing mate pair libraries. It enables the generation of long-range genomic information by creating DNA fragments with known spacing between the read pairs.
Automatically generated - may contain errors
Lab products found in correlation
Truseq pcr free kit, by Illumina (3 mentions)
Miseq machine, by Illumina (2 mentions)
Genelute bacterial genomic dna kit, by Merck Group (2 mentions)
Miseq with miseq reagent kit v3 2x301 bp, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Truseq rna access library prep kit, by Illumina (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Hiseq x system, by Illumina (1 mentions)
Nebnext multiplex oligos, by New England Biolabs (1 mentions)
Hiseq 1500, by Illumina (1 mentions)
Nucleobond buffer set 3, by Takara Bio (1 mentions)
Nebnext ultra dna library prep kit, by New England Biolabs (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
S2 sonicator, by Covaris (1 mentions)
Nextera xt kit, by Illumina (1 mentions)
Miseq system, by Illumina (1 mentions)
Miseq with miseq reagent kit v3, by Illumina (1 mentions)
10 protocols using nextera mate pair kit
1
Tumor DNA and RNA Extraction and Sequencing
2
Genomic DNA Extraction and Sequencing of Aspergillus Species
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The genomic DNAs of A. viridinutans and A. pseudoviridinutans were extracted from a 2-d-old culture using phenol-chloroform and NucleoBond buffer set III (TaKaRa, Shiga, Japan). The DNA was fragmented in an S2 sonicator (Covaris, MA, USA), and then purified using a QIAquick gel extraction kit (Qiagen, CA, USA). A paired-end library was constructed using an NEBNext Ultra DNA Library Prep Kit (New England BioLabs, MA, USA) and NEBNext Multiplex Oligos (New England BioLabs) in accordance with the manufacturer's instructions. Mate-paired libraries with insert sizes of 3.5–4.5, 5–7, and 8–11 kb were generated using the gel selection-based protocol of the Nextera Mate Pair Kit (Illumina, San Diego, CA, USA) and a 0.6% agarose gel, in accordance with the manufacturer's instructions. The quality of the libraries was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing (100 bp) was performed by HiSeq 1500 (Illumina) using HiSeq reagent kit v1, in accordance with the manufacturer's instructions; 150-bp paired-end sequencing on a HiSeq X system (Illumina) was carried out by GENEWIZ (South Plainfield, NJ, USA).
3
Whole Genome Sequencing of Legionella Outbreak
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WGS was performed on all patient isolates related to the outbreak, patient isolates obtained in the same period of time from elsewhere in the province of Quebec (contemporary), a subset of environmental isolates, as well as isolates from a former outbreak in the same area in 1996 [41] . WGS was performed using the Nextera XT kit (Illumina, San Diego, CA) as by the manufacturer and the sequencing (2×250 nt) performed on a MiSeq system (Illumina). Mate pair sequencing was performed on the same system using Nextera Mate Pair kit (Illumina) and the associated gel-plus protocol. Sequence assembly was performed using the Ray assembler version 2.2.0 [42] (link). Reads for the sample corresponding to the best assembly of Legionella pneumophila strain Quebec 2012 can be found under project number PRJEB5317 (Sequence Read Archive hosted by EMBL-EBI) and contigs for the same sample are available under accession numbers CCAA010000001 to CCAA010000048.
4
Genomic DNA Isolation and Sequencing
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Genomic DNA of strain 75/02 was isolated with GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Cat.Num.: NA2100) according to the manufacturer's instructions. Sequencing library was generated using Illumina Nextera Mate Pair Kit (Cat.Num.: FC-132-1001) as per manufacturer's instructions. DNA sequencing was performed on an Illumina MiSeq machine using V2 sequencing chemistry. Mate-paired reads were processed following the manufacturer's recommendations (Data Processing of Nextera® Mate Paired Reads on Illumina Sequencing Platforms).
5
Paired-End and Mate-Pair Sequencing
6
Whole genome sequencing library preparation
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DNA was extracted from 0.5 ml sample aliquots using the FastDNA® SPIN Kit for Soil (MP Biomedicals). Paired-end sample libraries were prepared using the Nextera or TruSeq PCR free kits (Illumina Inc.). In addition, a mate-pair library was generated using the Nextera Mate-pair kit (Illumina Inc.) with the gel-free approach. The prepared libraries were sequenced using either an Illumina MiSeq with MiSeq Reagent Kit v3 (2 × 301 bp; Illumina Inc.) or on an Illumina HiSeq2000 using the TruSeq PE Cluster Kit v3-cBot-HS and TruSeq SBS kit v.3-HS sequencing kit (2 × 150 bp; Illumina Inc.). See Supplementary Table S1 for an overview of library preparation and sequencing statistics for each sample.
7
Paired-End and Mate-Pair Sequencing
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DNA was extracted using the PowerSoil DNA isolation kit (MoBio, Carlsbad, CA) or a CTAB-based extraction method31 (link). 1 µg of DNA was used to prepare paired-end sequencing libraries using the TruSeq PCR-free kits (Illumina, San Diego, CA, USA) following the manufactures recommendation except that the 550 bp protocol was used with 1 µg of input DNA. Mate-pair libraries were prepared using the Nextera Mate-pair kit (Illumina) using the gel-free approach. The prepared libraries were sequenced using an Illumina MiSeq with MiSeq Reagent Kit v3 (2x301 bp; Illumina).
8
Whole Genome Mate Pair Sequencing Protocol
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Mate Pair sequencing (MPSeq) tiles the whole genome with large spanning (∼3 kb) fragments to increase the probability of spanning a genomic breakpoint. The MP library was assembled WGA DNA, according to a previously published protocol [3 (link)] using the Illumina Nextera Mate Pair kit (Illumina, CA; FC-132-1001). The MPSeq library was sequenced in half a lane of an Illumina flow cell and sequenced to 101×2 paired-end reads on an Illumina HiSeq2000. Base calling was carried out using Illumina Pipeline v1.5.
9
Mate-Pair Library Preparation for Genome Sequencing
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Mate-pair libraries were prepared using Nextera mate-pair kit following the manufacturers’ instructions (Illumina, San Diego, CA, USA). The subjects were investigated with the gel-free protocol where 1 μg of genomic DNA was fragmented using an enzymatic method generating fragments in the range of 2–15 kb. The final library was subjected to 2x100 bases paired-end sequencing on an Illumina HiSeq2500 sequencing platform.
10
E. coli T22 Genomic DNA Isolation and Sequencing
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Genomic DNA of E. coli T22 was isolated with GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich) according to the manufacturer's instructions. Clone library was generated using Illumina Nextera Mate Pair Kit (Cat.Num.: FC-132-1001) per the manufacturer's instructions. Re-sequencing was performed on an Illumina MiSeq machine using V2 sequencing chemistry. Mate-paired reads were pre-processed following the manufacturer's recommendations (Data Processing of Nextera® Mate Pair Reads on Illumina Sequencing Platforms).
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