Qiaseq 1 step amplicon library kit

Manufactured by Qiagen

The QIAseq 1-Step Amplicon Library Kit is a lab equipment product designed for preparing amplicon libraries for next-generation sequencing. The kit provides a streamlined workflow for generating sequencing-ready libraries from PCR amplicons.

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Lab products found in correlation

Generead dnaseq custom builder tool, by Qiagen (1 mentions) Kapa library quantification kit, by Roche (1 mentions) Nextseq, by Illumina (1 mentions) Ampure bead, by Beckman Coulter (1 mentions) Qiaseq library quant assay kit, by Qiagen (1 mentions) Phix control v3 library, by Illumina (1 mentions) Fragment analyzer, by Agilent Technologies (1 mentions) Agencourt ampure xp beads, by Thermo Fisher Scientific (1 mentions) Iseq100 cartridge, by Illumina (1 mentions) Local run manager software, by Illumina (1 mentions) Multiplex pcr kit, by Qiagen (1 mentions) Exosap it reagent, by Thermo Fisher Scientific (1 mentions) Hotstartaq master mix, by Qiagen (1 mentions)

4 protocols using qiaseq 1 step amplicon library kit

Customized Gene Panels for NGS Analysis

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We customized 2 different gene panels using Qiagen’s GeneRead DNAseq Custom Builder tool. The list of genes and its coverage regions are listed in Supplementary Tables 3, 4. Multiplex PCR was performed using GeneRead HotStar Taq DNA polymerase and four primer pools with a total of 80 ng of tumor or CTC DNA and 20ng of cell-free plasma DNA. The amplicons were pooled together and cleaned using AMPure beads (Beckman Coulter). The PCR-enriched DNA was subjected to next-generation sequencing library construction using QIAseq 1-Step Amplicon Library Kit (Qiagen). Each library was barcoded with a unique index and quantified using KAPA Library Quantification kit (Kapa Biosystems). Equal amounts of individual libraries were pooled together for a 150bp paired-end sequencing run on the Nextseq (Illumina) platform.

Kong S.L., Liu X., Tan S.J., Tai J.A., Phua L.Y., Poh H.M., Yeo T., Chua Y.W., Haw Y.X., Ling W.H., Ng R.C., Tan T.J., Loh K.W., Tan D.S., Ng Q.S., Ang M.K., Toh C.K., Lee Y.F., Lim C.T., Lim T.K., Hillmer A.M., Yap Y.S, & Lim W.T. (2021). Complementary Sequential Circulating Tumor Cell (CTC) and Cell-Free Tumor DNA (ctDNA) Profiling Reveals Metastatic Heterogeneity and Genomic Changes in Lung Cancer and Breast Cancer. Frontiers in Oncology, 11, 698551.

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Microsatellite Amplicon Sequencing Protocol

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PCR (20 ng of DNA) and LT-RPA (5 ng of DNA, 10.5 mM of Mg(OAc)₂ and 40 min incubation at 32°C) amplicons of HT17, NR24, CAT25, BAT26, D2S123, D18S61, D12ATA63, REN and HPRTII microsatellites (shorter primers were used for REN and HPRTII and are indicated in Supplementary Table S1) obtained for each blood sample were purified using Beckman Coulter™ Agencourt AMPure XP beads (Thermo Fischer Scientific), quantified and pooled in equimolar ratio. 900 ng of each pool were used for the ligation of dual-indexed adapters using QIAseq 1-Step Amplicon Library Kit (Qiagen) and no supplemental PCR amplification of the libraries was required. Libraries were assessed for quality and quantity with a Fragment Analyzer (Agilent) and QIAseq™ Library Quant Assay Kit (Qiagen) respectively. 50 pM of the pooled libraries were deposited on an iSeq 100 cartridge (Illumina) together with 20% of 50 pM PhiX control v3 Library (Illumina). Amplicon sequencing was performed on an iSeq 100 using 151 cycles of paired-end sequencing. FastQ files were generated for each sample using Local Run Manager Software (Illumina) and the read counts and sequences of each microsatellite allele were obtained from the aligned reads using an in-house developed Python code.

Daunay A., Duval A., Baudrin L.G., Buhard O., Renault V., Deleuze J.F, & How-Kit A. (2019). Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer. Nucleic Acids Research, 47(21), e141.

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Targeted Genomic DNA Sequencing Pipeline

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Genomic DNA was extracted as described above. Then the target genes were amplified by multiplex PCR with QIAGEN Multiplex PCR Kit (QIAGEN, 206143) with designed primers (Supplementary Table 3A). PCR products were purified by ExoSAP-IT reagent (Affymetrix, Inc.). A DNA library was prepared using the QIAseq 1-Step Amplicon Library Kit (QIAGEN, 180415). Next generation DNA sequencing (NGS) was performed on the ABI 3730 High-Throughput DNA Sequencer (Genomics Core Facility, Northwestern University) with the read depth of 105,718X (range of 78,090-152,121X). Mutations and variations were analyzed by DNASTAR Lasergene 9 software. Three SV involving in tumorigenic genes were selected and analyzed. Primer pairs to cover the upstream and downstream genes were designed using Primer 6 software (Supplementary Table 3B). Fusion genes/genomic DNA fragments were amplified by PCR with HotStar Taq Master Mix (Qiagen, 203446), and purified by ExoSAP-IT reagent (Affymetrix, Inc.). DNA sequencing was performed on the ABI 3730 High-Throughput DNA Sequencer.

Ban Y., Fischer J.V., Maniar K.P., Guo H., Zeng C., Li Y., Zhang Q., Wang X., Zhang W., Bulun S.E, & Wei J.J. (2020). Whole-Genome Sequencing and Target Validation Analysis of Müllerian Adenosarcoma: A Tumor With Complex but Specific Genetic Alterations. Frontiers in Oncology, 10, 538.

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CRISPR Mutation Analysis by Deep Sequencing

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The larvae hatched from embryo injections of Chitinase and ProbP CRISPR construct were pooled together and genomic DNA was extracted. The PCR amplicons surrounding predicted cut sites were generated using primers (Table S2). The deep sequencing library was prepared using QIAseq 1-Step amplicon library kit (Qiagen, 180412) following the manufacturer's recommended protocol and purified amplicons were sequenced on Illumina MiSeq (Novogene Inc, Davis, CA).Data were analyzed using a published script (Kistler et al., 2015 (link)) available at https://github.com/bnmtthws/crispr_indel.

Sharma A., Pham M.N., Reyes J.B., Chana R., Yim W.C., Heu C.C., Kim D., Chaverra-Rodriguez D., Rasgon J.L., Harrell RA I.I., Nuss A.B, & Gulia-Nuss M. (2022). Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control. iScience, 25(3), 103781.

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