Kapa hyperprep kit

Manufactured by Covaris

Sourced in United States

The KAPA HyperPrep Kit is a DNA library preparation kit designed for next-generation sequencing applications. The kit provides a streamlined and automated workflow for the construction of sequencing-ready libraries from genomic DNA or amplicons. The kit includes reagents and protocols for DNA fragmentation, end-repair, A-tailing, and adapter ligation.

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Lab products found in correlation

Magna pure compact system, by Roche (1 mentions) Quantus fluorometer, by Promega (1 mentions) Kapa hyper prep kit, by Illumina (1 mentions) Agilent 4200 tapestation system, by Agilent Technologies (1 mentions) Me220, by Covaris (1 mentions) Ampure xp bead, by Beckman Coulter (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Hiseq 4000, by Illumina (1 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions) Sureselectxt human all exon v5, by Agilent Technologies (1 mentions) Agilent tapestation high sensitivity d1000 assay, by Agilent Technologies (1 mentions) Kapa hyper prep kit, by Roche (1 mentions)

4 protocols using kapa hyperprep kit

Automated Extraction and NGS of mtDNA

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The samples' DNA was extracted from WBC (white blood cells) via the automated MagNA Pure Compact System (Roche Life Science) based on magnetic-beads technology, and all sample preparation procedures were conducted in accordance with the standard Illumina protocol (Human mtDNA Genome). We obtained two long fragments when performing two PCR reactions for further sequencing with the target size of each PCR amplicon in the range of 7800-10400 bp for MTL1 primer pairs and 9500-12500 bp for MTL2, respectively. Subsequently, the quantity of each sample was assessed with Quantus Fluorometer TM (Promega Corp., USA). The NGS itself comprised four stages described in Kapa Hyper Prep Kit and Illumina platform (MiSeq, USA) protocols, namely, fragmentation, end-repair, A-tailing, and adaptor ligation. We first pooled two PCR-products according to the volumes presented in Kapa Hyper Prep Kit and utilized Covaris S220 to obtain fragments with a size range of 200-250 bp. Following fragmentation, each sample was quantified via the Agilent 4200 TapeStation system, which was also applied to assess the samples' quality coupled with Quantus Fluorometer. All libraries displayed a fragment size of ~350bp and yield ~1μg.

Shikov A.E., Tsay V.V., Fedyakov M.А., Eismont Y.A., Rudnik A., Urazov S.P., Scherbak S.G., & Glotov O.S. (2021). The application of Nanopore sequencing for variant calling on the human mitochondrial DNA. Biological Communications, 66(2).

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DNA Fragmentation Methods Comparison

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For the KAPA HyperPrep Kit, gDNA was sonicated to 150–200 bp fragments using a Covaris ME220 instrument. For the experiment comparing fragmentation methods and DNA sources, DNA was sonicated in microTUBE AFA Fiber Pre-Slit Snap-Cap 6 × 16 mm tubes at 10 ng/μl in TE buffer (10 mM Tris–HCl, 1 mM EDTA) for 225 s at peak power of 75 W, 25% duty factor and 1000 cycles per burst. For the VAF series experiment, DNA was sonicated prior to mixing the two genotypes, at 50 ng/μl in TE buffer in 8 microTUBE AFA Beads Strip V2 for 140 s at peak power of 50, 30% duty factor and 50 cycles per burst. After sonication, we purified DNA with 1.8× volume ratio of AMPure XP beads (Beckman Coulter) and eluted into IDTE.

Alekseenko A., Wang J., Barrett D, & Pelechano V. (2022). OPUSeq simplifies detection of low-frequency DNA variants and uncovers fragmentase-associated artifacts. NAR Genomics and Bioinformatics, 4(2), lqac048.

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High-Quality Genomic DNA Extraction and Sequencing

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Genomic DNA (gDNA) was extracted using the QIAGEN DNeasy Blood and Tissue Kit (QIAGEN) following the mammal’s instructions. The extracted gDNA was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen), and approximately, 500 ng gDNA was sheared into around 300 bp fragments using Covaris S220, subsequently for library construction with KAPA Hyper Prep Kit. The final cycle for library amplification was 4 to reduce PCR bias. The samples were sequenced on illumina HiSeq-4000 with 150 bp pair-end reads.

Fan X., Yang C., Li W., Bai X., Zhou X., Xie H., Wen L, & Tang F. (2021). SMOOTH-seq: single-cell genome sequencing of human cells on a third-generation sequencing platform. Genome Biology, 22, 195.

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FFPE DNA Library Preparation and Enrichment

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For DNA extracted from Formalin-fixed paraffin embedded (FFPE) tissue, adapter-ligated libraries were prepared using the KAPA HyperPrep Kit (KAPA Biosystems, Wilmington, MA, USA) followed by Agilent SureSelectXT capture enrichment according to the manufacturers’ protocols. Samples were normalised to 400 ng and sheared to 150–200 bp using a Covaris E220 (Covaris, Woburn, MA, USA), following the parameters outlined in the KAPA HyperPrep Kit for SureSelect Target Enrichment protocol. KAPA HyperPrep libraries were generated and amplified using 10, 11 or 12 pre-capture PCR cycles and subsequently enriched using either the Agilent custom Melanoma Driver Panel or SureSelectXT Human All Exon v5 capture library. The quality and fragment size distributions of the purified libraries were assessed using the Agilent TapeStation High Sensitivity D1000 Assay (Agilent Technologies).

Spain L., Coulton A., Lobon I., Rowan A., Schnidrig D., Shepherd S.T., Shum B., Byrne F., Goicoechea M., Piperni E., Au L., Edmonds K., Carlyle E., Hunter N., Renn A., Messiou C., Hughes P., Nobbs J., Foijer F., van den Bos H., Wardenaar R., Spierings D.C., Spencer C., Schmitt A.M., Tippu Z., Lingard K., Grostate L., Peat K., Kelly K., Sarker S., Vaughan S., Mangwende M., Terry L., Kelly D., Biano J., Murra A., Korteweg J., Lewis C., O'Flaherty M., Cattin A.L., Emmerich M., Gerard C.L., Pallikonda H.A., Lynch J., Mason R., Rogiers A., Xu H., Huebner A., McGranahan N., Al Bakir M., Murai J., Naceur-Lombardelli C., Borg E., Mitchison M., Moore D.A., Falzon M., Proctor I., Stamp G.W., Nye E.L., Young K., Furness A.J., Pickering L., Stewart R., Mahadeva U., Green A., Larkin J., Litchfield K., Swanton C., Jamal-Hanjani M, & Turajlic S. (2023). Late-Stage Metastatic Melanoma Emerges through a Diversity of Evolutionary Pathways. Cancer Discovery, 13(6), 1364-1385.

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