Nextera chemistry

Manufactured by Illumina

Sourced in Sweden, United States

Nextera chemistry is a library preparation method developed by Illumina. It is used to generate sequencing-ready libraries from DNA samples. The core function of Nextera chemistry is to fragment and tag DNA samples in a single step, enabling efficient library preparation for next-generation sequencing.

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Lab products found in correlation

Hiseq 2500, by Illumina (2 mentions) Hiseq platform, by Illumina (1 mentions) Agilent sure select focused exome, by Agilent Technologies (1 mentions) Hiseq sequencer, by Illumina (1 mentions) Spark 10m, by Tecan (1 mentions) Quant ittm dsdna high sensitivity assay kit, by Tecan (1 mentions) Ez1 dna tissue kit, by Qiagen (1 mentions) Miseq sequencer, by Illumina (1 mentions) Wizard dna purification kit, by Promega (1 mentions) Clc genomics workbench software version 6, by Qiagen (1 mentions)

Spelling variants of this product

Nextera XT chemistry (8 citations) Nextera XT DNA chemistry (2 citations)

5 protocols using nextera chemistry

Exome Sequencing Protocol for Variant Identification

Cited 1 time

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Genomic DNA was extracted from peripheral blood lymphocytes. Focused exome sequencing was performed using the Agilent Sure Select Focused Exome (Agilent) according to the manufacturer’s protocol. WES was performed using Nextera chemistry (Illumina Inc). Sequencing was performed using the HiSeq platform (Illumina).
The Illumina fastq sequencing data were mapped to the human reference assembly, hg19 (GRCh37; UCSC genome browser) by Novoalign Software (Novocraft Inc). After removal of PCR duplicates (Picard) and reads without a unique mapping location, variants were extracted using the Maq model in SAMtools and outputted by the following criteria: consensus quality > 30, SNP quality > 30 and root mean square mapping quality > 30. These variant calls were then annotated using Annovar software (Wang et al., 2010 (link)).

Lynch D.S., Rodrigues Brandão de Paiva A., Zhang W.J., Bugiardini E., Freua F., Tavares Lucato L., Macedo-Souza L.I., Lakshmanan R., Kinsella J.A., Merwick A., Rossor A.M., Bajaj N., Herron B., McMonagle P., Morrison P.J., Hughes D., Pittman A., Laurà M., Reilly M.M., Warren J.D., Mummery C.J., Schott J.M., Adams M., Fox N.C., Murphy E., Davagnanam I., Kok F., Chataway J, & Houlden H. (2017). Clinical and genetic characterization of leukoencephalopathies in adults. Brain, 140(5), 1204-1211.

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Whole Genome Sequencing of Bacterial Isolates

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Whole genome sequencing (WGS) for available isolates (N = 262) was performed by using MiSeq or HiSeq sequencers (Illumina) as part of different projects at THL (N = 58), at Institute National de Saude Doutor Ricardo Jorge (INSA), Lisbon, Portugal (N = 44), and at SciLifeLab, Stockholm, Sweden (N = 160). Bacterial DNA extraction and genomic library preparation using Nextera chemistry (Illumina) were performed by using methods and procedures in each laboratory as previously described [28 , 29 (link)].
The WGS data analysis was performed at Karolinska Institutet, Sweden. The sequencing reads were quality-control processed and quality evaluated with QCtool pipeline (https://github.com/mtruglio/QCtool). The processed reads were assembled de novo with SPAdes (version: 3.12.0) in “careful mode” [30 (link)]. The assemblies were annotated with Prokka (version 1.11) [31 (link)].

Ylinen E., Salmenlinna S., Halkilahti J., Jahnukainen T., Korhonen L., Virkkala T., Rimhanen-Finne R., Nuutinen M., Kataja J., Arikoski P., Linkosalo L., Bai X., Matussek A., Jalanko H, & Saxén H. (2020). Hemolytic uremic syndrome caused by Shiga toxin–producing Escherichia coli in children: incidence, risk factors, and clinical outcome. Pediatric Nephrology (Berlin, Germany), 35(9), 1749-1759.

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Nextera DNA Library Preparation

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The DNA preparations were quantified using Quant-iTTM dsDNA High-Sensitivity Assay Kit and Tecan Spark 10 M plate reader. In total 1 μl of DNA (∼0.5–2.0 ng/μl) was used in the tagmentation reaction using Nextera chemistry (Illumina) to yield fragments >150 bp. The tagmented library underwent eleven cycles of PCR with single-end indexed primers (IDT Technologies) followed by purification using Seramag beads according to the protocol. The library was quantified using Quant-iTTM dsDNA High-Sensitivity Assay Kit and Tecan Spark 10 M plate reader and pair-end (2 × 101 bp) sequenced to a depth of 100,000–1,000,000 reads per sample on a HiSeq 2500 Illumina sequencer. Base calling and demultiplexing was done using bcl2fastq v1.87, without allowing any mismatch in the index sequence.

Dyrdak R., Mastafa M., Hodcroft E.B., Neher R.A, & Albert J. (2019). Intra- and interpatient evolution of enterovirus D68 analyzed by whole-genome deep sequencing. Virus Evolution, 5(1), vez007.

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Whole Genome Sequencing of C. difficile

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WGS was performed on 246 isolates. C. difficile isolates were grown anaerobically and genomic DNA was extracted after lysing the bacteria, using the EZ1 DNA Tissue kit (Qiagen, Hilden, Germany). WGS was performed at the SciLifeLab (Solna, Sweden). One µl of normalized genomic DNA (1.5 ng/µl) was used in the tagmentation reaction using Nextera chemistry (Illumina Inc., San Diego, CA) and pair-end (2 × 101 bp) sequenced to average 2–3 M read pairs on HiSeq 2500 (Illumina Inc., San Diego, CA).
Analysis of obtained sequences was performed by core genome MLST (cgMLST) based on 2583 alleles in SeqSphere+ using SKESA assembler v.2.3.0 (Ridom GmbH, Münster, Germany). The cluster threshold was set to ≤6 genes difference between isolates [22 (link)].

Magnusson C., Mernelius S., Bengnér M., Norén T., Serrander L., Forshell S, & Matussek A. (2022). Characterization of a Clostridioides difficile outbreak caused by PCR ribotype 046, associated with increased mortality. Emerging Microbes & Infections, 11(1), 850-859.

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Whole Genome Sequencing of Clinical and Environmental Isolates

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Whole genome shotgun sequencing was performed on 15 strains recovered from clinical and environmental samples in Israel. High molecular weight and quality DNA was extracted using the Wizard DNA purification kit (Promega, Madison, WI, United States). Sequencing libraries were prepared using the Nextera chemistry (Illumina Inc., San Diego, California, United States) for a 250 bp paired-end sequencing run on an Illumina MiSeq sequencer. Samples were sequenced to aim for a minimum coverage of 75-fold using Illumina's recommended standard protocols. All generated raw reads were submitted to the European Nucleotide Archive (ENA) (http://www.ebi.ac.uk/ena/) of the European Bioinformatics Institute under the study accession number PRJEB7140. After sequencing, the reads were quality-trimmed using the CLC Genomics Workbench software version 6.0 (CLC bio, Aarhus, Denmark) and then assembled de novo using CLC Genomics Workbench with default settings. The resulting assembly files were exported as ACE files and imported into SeqSphere + software version 2.1 (Ridom GmbH, Germany).

Moran-Gilad J., Prior K., Yakunin E., Harrison T.G., Underwood A., Lazarovitch T., Valinsky L., Luck C., Krux F., Agmon V., Grotto I, & Harmsen D. (2015). Design and application of a core genome multilocus sequence typing scheme for investigation of Legionnaires' disease incidents. Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 20(28).

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