Kapa library quantification kit

Manufactured by Illumina

Sourced in United States, Switzerland, Germany

The KAPA Library Quantification Kit is a qPCR-based assay used to accurately quantify libraries for sequencing on Illumina platforms. It provides a fast and reliable method to determine the concentration of DNA libraries, ensuring optimal cluster densities and sequencing performance.

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Library Quantification Kit (29 citations) KAPA Library Quantification Kits for Illumina sequencing platforms (10 citations) KAPA Library Quantification Kits for Illumina platforms (10 citations) KAPA Library Quantification Kits Illumina® Platforms (4 citations)

146 protocols using kapa library quantification kit

Sequencing Targeted CRISPR Genomic Edits

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PCR of genomic DNA and cDNA from muscles was performed using primers (listed in Supplementary Table S1) designed against the respective target region. A second round of PCR was used to add Illumina flowcell binding sequences and experiment-specific barcodes on the 5′ end of the primer sequence (Supplementary Table S1). Before sequencing, DNA libraries were analyzed using a Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent). Library concentration was then determined by qPCR using a KAPA Library Quantification Kit for Illumina platforms. Library concentration was then determined by qPCR using a KAPA Library Quantification Kit for Illumina platforms. The resulting PCR products were pooled and sequenced with 300 bp paired-end reads on an Illumina MiSeq instrument. Samples were demultiplexed according to assigned barcode sequences. FASTQ format data was analyzed using the CRISPResso software. The alignment of reads at the cleavage site was further analyzed and regrouped in the nonedited, single-nucleotide insertion (+1N), in frame insertion, out of frame insertion, and deletion groups.

Amoasii L., Li H., Zhang Y., Min Y.L., Sanchez-Ortiz E., Shelton J.M., Long C., Mireault A.A., Bhattacharyya S., McAnally J.R., Bassel-Duby R, & Olson E.N. (2019). In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse. Nature Communications, 10, 4537.

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Sequencing of Transcriptome Libraries

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One μg of high-quality total RNA was used to construct 120 cDNA libraries, using KAPA Stranded mRNA-Seq Kit (Roche, Switzerland), according to the manufacturer’s instructions. Each library was barcoded using SeqCap Adapter kit A and B (Roche NimbleGen, WI). Tapestation 2200 (Agilent) was used to confirm the final size (250–280 bps), using High Sensitivity D1000 ScreenTape kit (Agilent). KAPA Library Quantification kit—Illumina (Roche, Switzerland) was used to quantify the libraries on LightCycler 480 (Roche). Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) was used for sequencing, with paired end runs of 2 × 50 bps. Base calling and quality control were checked by Illumina RTA v1.13 (Illumina) sequence analysis pipeline. The original sequencing datasets have been deposited in the European Nucleotide Archive (ENA) with the accession number PRJEB32438.

Bianchi D., Ricciardi V., Pozzoli C., Grossi D., Caramanico L., Pindo M., Stefani E., Cestaro A., Brancadoro L, & De Lorenzis G. (2023). Physiological and Transcriptomic Evaluation of Drought Effect on Own-Rooted and Grafted Grapevine Rootstock (1103P and 101-14MGt). Plants, 12(5), 1080.

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Single-cell RNA-seq and ATAC-seq protocol

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The final library was quantified using the KAPA library quantification kit for Illumina platforms (KK4873, Roche, Indianapolis, IN). For gene expression libraries, sequencing was performed on a NextSeq 550 using high output kit or a HiSeq 4000 using SBS reagents (Illumina, Inc., San Diego, CA), with the following settings: Read 1 28 cycles, I7 index 8 cycles, Read 2 91 cycles. FASTQ files were generated from the Illumina sequencer Base Call files (bcl files) using 10x Genomics CellRanger mkfastq (version 3.1.0) software. Gene expression counts were generated by aligning reads to the Rnor 6.0 genome (NCBI: GCA_000001895.4) using CellRanger count software (v.3.1.0) (10x Genomics). After quality trimming, all fastq files belonging to a single GEM well but from different sequencing runs were pooled and counted together. As nuclear transcripts were unspliced, only reads mapped to pre-mRNA were counted. For snATAC-seq libraries, sequencing was performed on an Illumina NextSeq 550 using high output kit (150 cycles) (Illumina, Inc., San Diego, CA), with the following settings: Read 1 50 cycles, I7 index 8 cycles, I5 index 16 cycles, Read 2 50 cycles. FASTQ files were generated from bcl files using 10x Genomics CellRanger-atac mkfastq (version 1.2.0) software.

Chen Z., Chen W., Li Y., Moos M J.r., Xiao D, & Wang C. (2022). Single-nucleus chromatin accessibility and RNA sequencing reveal impaired brain development in prenatally e-cigarette exposed neonatal rats. iScience, 25(8), 104686.

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Amplification and sequencing of bacterial 16S rRNA and fungal ITS1 regions

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Total extracted DNA was used for high throughput sequencing (MiSeq platform, Illumina, San Diego, CA, USA) of the bacterial 16S rRNA gene or fungal ITS1 region. We amplified the V3–V4 region of 16S rRNA gene using degenerate barcoded primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), and the ITS1 region using degenerate fungal primers ITS1-5.8Sfw (5′-CTGTAAAAGTCGTAACAAGGTTTC-3′) and ITS1-5.8Srv (5′-AAGTTCAAAGAYTCGATGATTCAC-3′). PCR amplifications (KAPA 2G Robust Hot Start DNA Polymerase, Kapa Biosystems, Hoffmann-La Roche, Switzerland) were carried out with 25 or 27 cycles, respectively. PCR products were purified and normalized with the SequalPrep™ Normalization Plate Kit (Thermo Fisher Scientific, Waltham, MA, USA). Triplicates of the amplicons were pooled, gel eluted (NucleoSpin gel clean-up, Macherey-Nagel, Düren, Germany), and ligated with sequencing adapters (TruSeq DNA PCR-free LT Sample Preparation Kit, Illumina; KAPA HyperPlus kit, Kapa Biosystems, Hoffmann-La Roche, Switzerland). Amplicon libraries were pooled in equimolar concentrations, validated by the KAPA Library Quantification Kit (Illumina, San Diego, CA, USA), and sequenced on the MiSeq platform using the 2 × 300 bp kit at the CEITEC Genomics Core Facility (CEITEC, Masaryk University, Brno, Czech Republic).

Stehlikova Z., Tlaskal V., Galanova N., Roubalova R., Kreisinger J., Dvorak J., Prochazkova P., Kostovcikova K., Bartova J., Libanska M., Cermakova R., Schierova D., Fassmann A., Borilova Linhartova P., Coufal S., Kverka M., Izakovicova-Holla L., Petanova J., Tlaskalova-Hogenova H, & Jiraskova Zakostelska Z. (2019). Oral Microbiota Composition and Antimicrobial Antibody Response in Patients with Recurrent Aphthous Stomatitis. Microorganisms, 7(12), 636.

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RNA-seq Analysis of NS vs HAL Mice

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RNA from F1 mice (NS vs HAL; n = 6 each) was subjected to RNA-seq. The RNA integrity number was calculated by an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA) and an Agilent RNA 6000 Nano Kit (Agilent Technologies Inc.) (supplementary Table 1). Subsequently, 200 ng total RNA from each sample was used for RNA-seq library preparation. An RNA-seq library of each sample was created using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Indianapolis, IN, USA) according to the manufacturer’s protocol. The quality of the average size (340–380 bp) was validated using an Agilent DNA1000 kit and Agilent 2100 Bioanalyzer (Agilent Technologies Inc.). The amount was determined using qPCR with the Kapa Library Quantification Kit (Illumina). The MiSeq Reagent kit V3 on a MiSeq system was used for sequencing (Illumina) based on pair-end reads (75 bp) according to the manufacturer’s instructions. The running cycle was set to 150 cycles.

Yoshino Y., Kumon H., Shimokawa T., Yano H., Ochi S., Funahashi Y., Iga J.I., Matsuda S., Tanaka J, & Ueno S.I. (2022). Impact of Gestational Haloperidol Exposure on miR-137-3p and Nr3c1 mRNA Expression in Hippocampus of Offspring Mice. International Journal of Neuropsychopharmacology, 25(10), 853-862.

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Single-Cell RNA-Seq of TILs from HGSOC

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TRM-like and re-circulating CD8⁺ TILs were sorted form 7 different HGSOCs. Total RNA was isolated from sorted cells using RNA isolation kit (Qiagen) and analyzed for RINe. Next gen RNA sequencing was performed by the Moffitt Cancer Center Molecular Genomics facility.
One nanogram of RNA was processed for RNA-seq using the NuGEN Ovation SoLo RNA-Seq system. This low-input kit generates strand-specific libraries rRNA depleted libraries using an inline probe-based depletion step referred to as AnyDeplete. The cDNA was generated and libraries were prepared according to the manufacturer’s protocol, including the optional inline DNase digestion step. Final libraries were quantitated with the Kapa Library Quantification kit and were sequenced on the Illumina NextSeq 500 in order to generate 8-10 million pairs of 75-base paired-end reads per sample.

Anadon C.M., Yu X., Hanggi K., Biswas S., Chaurio R., Martin A., Payne K.K., Mandal G., Innamarato P., Harro C.M., Mine J., Sprenger K., Cortina C., Powers J.J., Costich T.L., Perez B.A., Gatenbee C.D., Prabhakaran S., Marchion D., Heemskerk M.H., Curiel T.J., Anderson A.R., Wenham R.M., Rodriguez P.C, & Conejo-Garcia J.R. (2022). Ovarian cancer immunogenicity is governed by a narrow subset of progenitor tissue-resident memory T-cells. Cancer cell, 40(5), 545-557.e13.

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Targeted Resequencing of Disease-Associated Region

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Exon resequencing and sequencing of an expanded GAA repeat allele were performed using a standard Sanger sequencing methodology, using Bigdye v3.1 chemistry. Sequencing products were separated on ABI3100 genetic analysers and data analysed using the Staden Gap4 software package.
Libraries for targeted resequencing of the disease-associated region were prepared using a custom SureSelect XT target enrichment kit (Agilent). Capture probes (baits) were design using the online tool e-array (https://earray.chem.agilent.com/earray/). Final libraries were quantified using the Kapa library quantification kit, and sequencing was performed at the Wellcome Trust Centre for Human Genetics, University of Oxford, on an Illumina HiSeq 2000. A 20 Gb dataset of 51 bp paired-end reads was produced. Reads were aligned to the CanFam2 genome sequence using BWA (Li and Durbin 2009 (link)), and SNP and INDEL calls made using GATK (McKenna et al. 2010 (link)). Read alignments were visualised using the Integrative Genome Viewer (IGV) (Thorvaldsdottir et al. 2013 (link)).

Forman O.P., De Risio L., Matiasek K., Platt S, & Mellersh C. (2014). Spinocerebellar ataxia in the Italian Spinone dog is associated with an intronic GAA repeat expansion in ITPR1. Mammalian Genome, 26(1), 108-117.

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Whole-Genome Sequencing of PARK2 Carriers

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To dissect the novel GWAS signal associated with AAO identified in the Spanish population, we performed whole-genome sequencing (WGS) analyses in 5 of 37 homozygous carriers of the PARK2 signal. DNA concentration was determined by Qubit fluorescence and normalized to 20 ng/uL. One microgram of total genomic DNA was sheared to a target size of 450 base pairs (bp) using the Covaris LE220 ultrasonicator. Library preparation was achieved using the TruSeq DNA PCR-Free High Throughput Library Prep Kit and IDT for Illumina TruSeq DNA UD Indexes (96 Indexes, 96 Samples). Sequencing libraries were assessed for size distribution, absence of free adapters, and adapter dimers on a Fragment Analyzer. Library quantitation was performed by quantitative polymerase chain reaction using the KAPA Library Quantification Kit subsequent normalization to 4 nM. Libraries were clustered on v2.5 flowcell using the Illumina cBot 2 System before sequencing on the Illumina HiSeq X System using paired-end 150-bp reads. BCL files processed with alignment by ISAAC on HAS 2.2 and BAMs were used for QC assessment of mean coverage, percent duplicates, percent bases >20× coverage, and percent noise sites.

Bandres-Ciga S., Ahmed S., Sabir M.S., Blauwendraat C., Adarmes-Gómez A.D., Bernal-Bernal I., Bonilla-Toribio M., Buiza-Rueda D., Carrillo F., Carrión-Claro M., Gómez-Garre P., Jesús S., Labrador-Espinosa M.A., Macias D., Méndez-del-Barrio C., Periñán-Tocino T., Tejera-Parrado C., Vargas-González L., Diez-Fairen M., Alvarez I., Tartari J.P., Buongiorno M., Aguilar M., Gorostidi A., Bergareche J.A., Mondragon E., Vinagre-Aragon A., Croitoru I., Ruiz-Martínez J., Dols-Icardo O., Kulisevsky J., Marín-Lahoz J., Pagonabarraga J., Pascual-Sedano B., Ezquerra M., Cámara A., Compta Y., Fernández M., Fernández-Santiago R., Muñoz E., Tolosa E., Valldeoriola F., Gonzalez-Aramburu I., Sanchez Rodriguez A., Sierra M., Menéndez-González M., Blazquez M., Garcia C., Suarez-San Martin E., García-Ruiz P., Martínez-Castrillo J.C., Vela-Desojo L., Ruz C., Barrero F.J., Escamilla-Sevilla F., Mínguez-Castellanos A., Cerdan D., Tabernero C., Gomez Heredia M.J., Perez Errazquin F., Romero-Acebal M., Feliz C., Lopez-Sendon J.L., Mata M., Martínez Torres I., Kim J.J., Dalgard C.L., Brooks J., Saez-Atienzar S., Gibbs J.R., Jorda R., Botia J.A., Bonet-Ponce L., Morrison K.E., Clarke C., Tan M., Morris H., Edsall C., Hernandez D., Simon-Sanchez J., Nalls M.A., Scholz S.W., Jimenez-Escrig A., Duarte J., Vives F., Duran R., Hoenicka J., Alvarez V., Infante J., Marti M.J., Clarimón J., López de Munain A., Pastor P., Mir P, & Singleton A. (2019). The Genetic Architecture of Parkinson Disease in Spain: Characterizing Population-Specific Risk, Differential Haplotype Structures, and Providing Etiologic Insight. Movement disorders : official journal of the Movement Disorder Society, 34(12), 1851-1863.

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Transcriptome Analysis of Drosophila Eye and Wing Discs

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Per replicate and genotype 40 eye antennal discs or 30 wing discs from third instar larvae were dissected. RNA was extracted via peqGold MicroSpin Total RNA Kit. Library preparation and RNA-Seq were carried out according to the NEBNext Ultra RNA Library Prep protocol, the Illumina HiSeq 1000 System User Guide, and the KAPA Library Quantification Kit—Illumina/ABI Prism User Guide. Library preparation and RNA-Seq were performed at the Genomics Core Facility “KFB—Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany).

Rass M., Gizler L., Bayersdorfer F., Irlbeck C., Schramm M, & Schneuwly S. (2022). The Drosophila functional Smad suppressing element fuss, a homologue of the human Skor genes, retains pro-oncogenic properties of the Ski/Sno family. PLoS ONE, 17(1), e0262360.

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RNA Sequencing Library Preparation

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RNA samples were assessed for quality with an Advanced Analytics fragment analyzer (high-sensitivity RNA analysis kit no. DNF-491) and quantity with a Qubit RNA quantification kit (Qubit RNA HS assay kit no. Q32855). Samples were used for library builds with the Clontech Smart-Seq V4 Ultra Low Input RNA kit from TaKaRa (catalog no. 634890). Upon library build completion, samples had quality and average fragment size assessed with the Advanced Analytics fragment analyzer (high-sensitivity NGS analysis kit no. DNF-486). Quantity was assessed with an Illumina universal adaptor-specific qPCR kit from Kapa Biosystems (Kapa library quantification kit for Illumina NGS no. KK4824). After final library quality control (QC) was completed, samples were equimolar pooled and clustered for sequencing on the NextSeq500 machine. The paired-end sequencing run was performed using Illumina HighSeq2500 run chemistry (HiSeq2500 high-output 2x100PE 8-lane run, 200 total cycles, FC-401-3001).

Merritt E.F., Johnson H.J., Wong Z.S., Buntzman A.S., Conklin A.C., Cabral C.M., Romanoski C.E., Boyle J.P, & Koshy A.A. (2020). Transcriptional Profiling Suggests T Cells Cluster around Neurons Injected with Toxoplasma gondii Proteins. mSphere, 5(5), e00538-20.

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