Nextera xt dna library preparation protocol

Manufactured by Illumina

Sourced in United States

Nextera XT DNA Library preparation protocol is a laboratory equipment product by Illumina. It is a streamlined library preparation method that generates high-quality sequencing libraries from low amounts of input DNA. The protocol uses a tagmentation process to simultaneously fragment and tag the DNA with adapter sequences in a single step.

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Lab products found in correlation

Qubit fluorometer, by Thermo Fisher Scientific (4 mentions) Nanodrop, by Thermo Fisher Scientific (4 mentions) Yeast genomic dna purification kit, by Avantor (2 mentions) Nebnext ultra 2 dna library prep protocol, by New England Biolabs (2 mentions) Rnase a, by Avantor (2 mentions) Hiseq x10, by Illumina (2 mentions) Hiseq 3000, by Illumina (2 mentions) Genfind v3, by Beckman Coulter (1 mentions) Native barcoding kit exp nbd103, by Oxford Nanopore (1 mentions) Hiseq 2500, by Illumina (1 mentions) Magna pure 96 system, by Roche (1 mentions) Midi kit, by Qiagen (1 mentions) Short read sequencing, by Illumina (1 mentions) Miseq sequencer, by Illumina (1 mentions) Miseq sequencing, by Illumina (1 mentions) Kapa hifi hotstart readymix, by Roche (1 mentions) Quantus fluorometer, by Promega (1 mentions) Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Magjet ngs cleanup kit, by Thermo Fisher Scientific (1 mentions) High capacity cdna rt kit, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Nextera XT DNA Library Preparation Nextera DNA library preparation protocol Nextera XT library preparation Nextera Library Preparation protocol Nextera XT DNA protocol Nextera XT protocol Nextera XT library Nextera XT DNA Nextera XT Nextera protocol

6 protocols using nextera xt dna library preparation protocol

Closed Bacterial Genomes via Short- and Long-Read Sequencing

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DNA was extracted using the MagNAPure 96 system (Roche Applied Science, Manheim, Germany) and sequencing libraries were prepared using Nextera XT DNA Library preparation protocol (Illumina, San Diego, CA, USA). Paired-end reads (2 × 150, 2 × 250 or 2 × 300 bp) were generated for all isolates using Illumina MiSeq with v3 chemistry. For isolates where only FASTQ files were received, sequencing was performed on an Illumina HiSeq 2500 at Eurofins Genomics (Eurofins Genomics Europe, Konstanz, Germany) generating 2 × 125 bp paired-end reads.
To achieve closed genomes, selected isolates were also long-read sequenced. DNA was extracted manually using the Beckman Coulter Life Science GenFind v3 kit (C34881) according to the supplemental protocol ‘DNA extraction from Bacteria using GenFind v3’ (Beckman Coulter, Brea, CA, USA). DNA libraries were prepared using the 1D Ligation sequencing kit (SQK-LSK108) and the Native barcoding kit (EXP-NBD103) [Oxford Nanopore Technologies (ONT), Oxford, UK] according to the ONT protocol ‘native barcoding genomic DNA’ or ‘genomic DNA by ligation’ without shearing to maximize the sequencing read length. Finally, libraries were loaded onto a R9.4.1 MinION flow cell (FLO-MIN106) or a R9.4.1 Flongle flow cell (FLO-FLG001) and sequenced on an ONT MinION Mk1B device (MIN-101B).

Fostervold A., Hetland M.A., Bakksjø R., Bernhoff E., Holt K.E., Samuelsen Ø., Simonsen G.S., Sundsfjord A., Wyres K.L, & Löhr I.H. (2021). A nationwide genomic study of clinical Klebsiella pneumoniae in Norway 2001–15: introduction and spread of ESBLs facilitated by clonal groups CG15 and CG307. Journal of Antimicrobial Chemotherapy, 77(3), 665-674.

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Yeast Genomic DNA Purification and Library Prep

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DNA was purified using the yeast genomic DNA purification kit (VWR). Briefly, cells were disrupted with sodium dodecyl sulphate (SDS) (VWR) at 65 °C. The pellet was then treated with ammonium acetate and RNase A (VWR). DNA was precipitated with isopropanol and stored in Tris-EDTA buffer at a pH of 8.0 (VWR). DNA purity was assessed via Nanodrop (Thermo Fisher Scientific) and 1% agarose gel electrophoresis. The DNA concentration was measured via Qubit fluorometer (Thermo Fisher Scientific). Initial library construction for the isolates entailed tagmentation via the Nextera XT DNA library preparation protocol (Illumina). Isolates were initially sequenced on a HiSeq 3000 (Illumina), generating 100bp paired reads. Samples with poor coverage resulting from this first method then underwent additional sequencing, with libraries generated in accordance with the NEBNext Ultra II DNA Library Prep protocol (New England Biolabs). Libraries were sequenced on a HiSeq X10 (Illumina), generating 150bp paired reads (minimum average coverage 10X).

Sephton-Clark P., Nguyen T., Hoa N.T., Ashton P., van Doorn H.R., Ly V.T., Le T, & Cuomo C.A. (2023). Impact of pathogen genetics on clinical phenotypes in a population of Talaromyces marneffei from Vietnam. bioRxiv.

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Whole-genome sequencing of carbapenem-resistant Klebsiella pneumoniae

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Five common carbapenemase genes (KPC, NDM, IMP, VIM, and OXA-48) were initially detected by PCR amplification and DNA sequencing (Gülmez et al., 2008 (link); Poirel et al., 2011 (link); Weiß et al., 2017 (link)). To fully understand the resistance genes among K. pneumoniae L204, in particular the carbapenemase, ESBL, and AmpC, we performed whole-genome sequencing analysis for this isolate. The genomic DNA of K. pneumoniae L204 was extracted by using a commercial Qiagen Midi kit (Qiagen, Hilden, Germany). The sequencing libraries were prepared using the Nextera XT DNA Library preparation protocol (Illumina, San Diego, CA, United States) and then the genomic DNA was subjected to Illumina (Illumina, San Diego, CA, United States) short-read sequencing (150 bp paired-end reads). Reads were trimmed with a sickle (GitHub) and were de novo assembled using SPAdes 3.12.0. Antimicrobial resistance genes analysis was performed using ResFinder 4.1¹ and further verified using NCBI BLAST and the annotation process was done using RAST version 2.0.²

Li S., Shen S., Ding L., Han R., Guo Y., Yin D., Guan M, & Hu F. (2022). First Report of blaCTX–M–167, blaSHV–1, and blaTEM–1B Carrying Klebsiella pneumonia Showing High-Level Resistance to Carbapenems. Frontiers in Microbiology, 13, 916304.

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Amplicon Sequencing of 18S rDNA

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After pooling, the samples were prepared for MiSeq™ Illumina sequencing following the Nextera XT DNA Library Preparation protocol (Illumina, USA) with the following modifications: a fragment (V4 region) of the 18S ribosomal (r) DNA was amplified using KAPA HiFi HotStartReadyMix (Kapa Biosystems, Inc., USA) and the following primer set: 528iF (GCG GTA ATT CCA GCT CCA A) and 964iR (ACTTT CGT TCT TGA TYR R) (Fadeev et al., 2018 (link)). The success of this amplicon PCR was confirmed with gel electrophoresis using 2 μL of the PCR product. If no bands were detected, the amplicon PCR was repeated with an increased template volume (up to 5 μL). If this still was not sufficient to detect the respective band, five additional cycles were added to the original program (eight samples). Before library normalization and pooling, the DNA concentration was once again measured using a Quantus Fluorometer (Promega, USA) and diluted accordingly. Amplicon sequencing was then performed on an Illumina MiSeq™ sequencer (Illumina, USA), and about 6.3 million 2 × 300 bp paired-end reads were produced in total.

Käse L., Kraberg A.C., Metfies K., Neuhaus S., Sprong P.A., Fuchs B.M., Boersma M, & Wiltshire K.H. (2020). Rapid succession drives spring community dynamics of small protists at Helgoland Roads, North Sea. Journal of Plankton Research, 42(3), 305-319.

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Viral RNA Sequencing Protocol

Cited 1 time

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Whole genome sequencing was
done as described previously.^{57 (link)} Briefly,
viral RNA from each passage of the evolution experiment was extracted
using the QIAamp Viral RNA Mini Kit (Qiagen, 52906). Viral mRNA was
isolated by poly-A pulldown with oligo d(T) 25 beads using the NEBNext
Poly(A) mRNA Magnetic Isolation Module (NEB, E7490L). The first strand
of cDNA was prepared using the High-Capacity cDNA RT Kit (ThermoFisher,
4368814), and the second strand was synthesized with the large Klenow
fragment of DNA polymerase 1 (NEB, M0210L). cDNA was purified with
the MinElute PCR purification kit (Qiagen, 28004). Tagmentation and
creation of the library was done using the Nextera XT DNA Library
Preparation Protocol (Illumina, FC-131–1096). Index adapters
used for sample identification were created by MBSU (Molecular Biology
Service Unit, University of Alberta, Edmonton, AB, Canada), and libraries
were cleaned up using the MagJET NGS Cleanup Kit (ThermoFisher, K2821).
Sample concentration was checked using the NanoDrop 8000 spectrophotometer
(ThermoFisher, ND-8000-GL), and sample purity was analyzed by the
Agilent 2100 Bioanalyzer G2938C (Marshall Scientific, AG-2100C) at
the MBSU. Equal amounts of DNA were combined and sent for sequencing
to the MBSU for Next Generation Sequencing on the MiSeq System (Illumina,
SY-410–1003).

Oraby A.K., Bilawchuk L., West F.G, & Marchant D.J. (2024). Structure-Based Discovery of Allosteric Inhibitors Targeting a New Druggable Site in the Respiratory Syncytial Virus Polymerase. ACS Omega, 9(20), 22213-22229.

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Genomic DNA Purification and Sequencing

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DNA was purified using the yeast genomic DNA purification kit (VWR). Briefly, cells were disrupted with sodium dodecyl sulphate (SDS) (VWR) at 65°C. The pellet was then treated with ammonium acetate and RNase A (VWR). DNA was precipitated with isopropanol and stored in Tris-EDTA buffer at a pH of 8.0 (VWR). DNA purity was assessed via Nanodrop (Thermo Fisher Scientific) and 1% agarose gel electrophoresis. The DNA concentration was measured via Qubit fluorometer (Thermo Fisher Scientific). Initial library construction for the isolates entailed tagmentation via the Nextera XT DNA library preparation protocol (Illumina). Isolates were initially sequenced on a HiSeq 3000 (Illumina), generating 100-bp paired reads. Samples with poor (<10X) or biased coverage resulting from this first method then underwent additional sequencing, with libraries generated in accordance with the NEBNext Ultra II DNA Library Prep protocol (New England Biolabs). Libraries were sequenced on a HiSeq X10 (Illumina), generating 150-bp paired reads (minimum coverage 10X per sample).

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