Xgen universal blocking oligos

Manufactured by Integrated DNA Technologies

XGen Universal blocking oligos are short DNA sequences designed to block non-specific primer binding during next-generation sequencing library preparation. These blocking oligos help reduce the formation of primer-dimers and off-target amplification, improving the efficiency and accuracy of sequencing experiments.

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Lab products found in correlation

Human cot 1 dna, by Thermo Fisher Scientific (7 mentions) Hiseq 4000 ngs platforms, by Illumina (4 mentions) Dynabeads m 270, by Thermo Fisher Scientific (4 mentions) Nimblegen seqcap ez hybridization and wash kit, by Roche (4 mentions) Kapa hyper prep kit, by Roche (3 mentions) Agencourt ampure xp bead, by Beckman Coulter (3 mentions) Kapa hifi hotstart readymix, by Roche (3 mentions) 2100 bioanalyzer, by Agilent Technologies (3 mentions) Kapa library quantification kit, by Roche (3 mentions) Ampure xp beads, by Roche (3 mentions) M220 instrument, by Roche (2 mentions) M220 instrument, by Covaris (1 mentions) Customized xgen lockdown probes panel, by Integrated DNA Technologies (1 mentions) Xgen lockdown probe panel, by Integrated DNA Technologies (1 mentions) Xgen lockdown probes, by Integrated DNA Technologies (1 mentions) Hiseq 4000ngs, by Illumina (1 mentions) Hiseq x ten platform, by Illumina (1 mentions) Xgen exome research panel v1, by Integrated DNA Technologies (1 mentions) Hiseq ngs platform, by Illumina (1 mentions) Nanodrop one, by Thermo Fisher Scientific (1 mentions)

7 protocols using xgen universal blocking oligos

Targeted Sequencing of Tumor Genomic DNA

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Extracted tumor genomic DNA was fragmented into 300 ~ 350 bp using Covaris M220 instrument (Covaris). Sequencing libraries were prepared with KAPA Hyper Prep kit (KAPA Biosystems) with optimized protocols. In brief, cfDNA or sheared tissue DNA were experienced with end-repairing, A-tailing, adapter ligation and size selection using Agencourt AMPure XP beads (Beckman Coulter). Libraries were then subjected to PCR amplification and purification before targeted enrichment.
Indexed DNA libraries were pooled up to 2 µg together with Human cot-1 DNA (Life Technologies) and xGen Universal blocking oligos (Integrated DNA Technologies) as blocking reagents. Customized xGen lockdown probes panel (Integrated DNA Technologies) were used to selectively enrich for 425 or 139 predefined genes. The enriched libraries were sequenced on Hiseq 4000 NGS platforms (Illumina) with 2 × 150 bp pair-end reads to coverage depths of at least 100x, 600x, 5000 × for blood, FFPE, and cfDNA, respectively.

Jin Y., Lin C., Shi X., He Q., Yan J., Yu X, & Chen M. (2022). Impact of clinical and molecular features on efficacy and outcome of patients with non-small cell lung cancer receiving second-line osimertinib. BMC Cancer, 22, 586.

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Comprehensive Cancer Gene Panel Sequencing

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cfDNA or fragmented genomic DNA (300~350 bp with Covaris M220 instrument) underwent sequencing library preparation using the KAPA Hyper Prep kit (KAPA Biosystems). In brief, DNA was experienced with end-repairing, A-tailing, adapter ligation, and size selection using Agencourt AMPure XP beads (Beckman Coulter) and then was amplified by PCR and purified.
Indexed DNA libraries were pooled up to 2 µg, together with Human cot-1 DNA (Life Technologies) and xGen Universal blocking oligos (Integrated DNA Technologies) as blocking reagents. A customized xGen lockdown probe panel (Integrated DNA Technologies) covering 425 predefined cancer-related genes was used to perform hybridization capture (Table S1). Enriched libraries were sequenced on Hiseq 4000 NGS platforms (Illumina) to target mean coverage depths of at least 100× for WBCs, 1000× for tissue- and bile pellet–derived DNA, and 5,000× for plasma- and bile supernatant–derived cfDNA.

Li Z., Liu Y., Fu J., Mugaanyi J., Yan J., Lu C, & Huang J. (2022). Bile is a reliable and valuable source to study cfDNA in biliary tract cancers. Frontiers in Oncology, 12, 961939.

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Cancer-Relevant Gene Capture and Sequencing Protocol

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Libraries with different indexes were pooled together in optimized ratios to reach up to 2 μg of total DNA. Human Cot-1 DNA (Life Technologies) and xGen universal blocking oligos (Integrated DNA Technologies) were added as blocking reagents to reduce non-specific hybridization. Customized xGen lockdown probes (Integrated DNA Technologies) targeting 5,804 exons of 382 cancer-relevant genes and 37 introns of 16 fusion genes were used for hybridization enrichment (Supplementary Table 1). The capture reaction was performed with NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche) and Dynabeads M-270 (Life Technologies) according to manufacturers’ protocols. Post-captured libraries were PCR amplified with Illumina p5 (5′ AAT GAT ACG GCG ACC ACC GA 3′) and p7 primers (5′ CAA GCA GAA GAC GGC ATA CGA GAT 3′) in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification with Agencourt AMPure XP beads, and quantified by qPCR method using KAPA Library Quantification kit (KAPA Biosystems). The size distribution of the library was analyzed by Bioanalyzer 2100 (Agilent Technologies). Target-enriched libraries were then sequenced on Illumina MiSeq or HiSeq4000 NGS platforms (Illumina) according to manufacturer's instructions. Targeted capture and sequencing performance of all the samples were summarized in Supplementary Table 3.

Du J., Wu X., Tong X., Wang X., Wei J., Yang Y., Chang Z., Mao Y., Shao Y.W, & Liu B. (2017). Circulating tumor DNA profiling by next generation sequencing reveals heterogeneity of crizotinib resistance mechanisms in a gastric cancer patient with MET amplification. Oncotarget, 8(16), 26281-26287.

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Whole-Exome Capture and Sequencing Protocol

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DNA libraries were subjected to whole-exome capture with xGen Exome Research Panel v1.0 (Integrated DNA Technologies), which spans a 39 Mb target region (19,396 genes) of the human genome and covers 51 Mb of end-to-end tiled space. Human Cot-1 DNA (Life Technologies) and xGen universal blocking oligos (Integrated DNA Technologies) were added as blocking reagents to reduce non-specific hybridization. The capture reaction was conducted with NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche) and Dynabeads M-270 (Life Technologies) according to manufacturer’s recommended protocol. The captured samples were sequenced on an Illumina HiSeq X-TEN platform with a paired-end run of 2×150 bp. The quality of each read was initially verified using the software embedded in the HiSeq X-TEN sequence. A FASTQ file was generated for each tested sample for sequence alignment and converted to a BAM file for further analysis.

Jiang T., Cheng R., Pan Y., Zhang H., He Y., Su C., Ren S, & Zhou C. (2020). Heterogeneity of neoantigen landscape between primary lesions and their matched metastases in lung cancer. Translational Lung Cancer Research, 9(2), 246-256.

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Targeted DNA Sequencing Library Preparation

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DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany. Purified DNA was qualified by Nanodrop One (Thermo Fisher, Waltham, MA) and quantified by Qubit 3.0 (Life Technologies, Singapore) using the dsDNA HS Assay Kit (Life Technologies, Eugene, OR) according to the manufacturer’s recommendations. Different libraries with unique indices were pooled together in desirable ratios for up to 2 μg of total library input. Human cot-1 DNA (Life Technologies, Carlsbad, CA) and xGen Universal blocking oligos (Integrated DNA Technologies, Coralville, IA) were added as blocking reagents. The capture reaction was performed with the NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche, Madison, WI) and Dynabeads M-270 (Life Technologies, Vilnius, Lithuania) with optimized manufacturers’ protocols. Captured libraries were on-beads amplified with Illumina p5 and p7 primers in KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Cape Town, South Africa). The post-capture amplified library was purified by Agencourt AMPure XP beads and quantified by qPCR using the KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by the Agilent Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA). The target-enriched library was then sequenced on HiSeq NGS platforms (Illumina, San Diego, CA) according to the manufacturer’s instructions.

Gan J., Huang Y., Liao J., Pang L, & Fang W. (2021). HER2 Amplification in Advanced NSCLC Patients After Progression on EGFR-TKI and Clinical Response to EGFR-TKI Plus Pyrotinib Combination Therapy. OncoTargets and therapy, 14, 5297-5307.

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Targeted Enrichment of Cancer Genes

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DNA libraries were subjected to polymerase chain reaction (PCR) amplification and purification before targeted enrichment. Libraries from different samples were marked with unique indices during library preparation and up to 2 μg of different libraries were pooled together for targeted enrichment. Human cot-1 DNA (Life Technologies) and xGen Universal Blocking Oligos (Integrated DNA Technologies) were added to block nonspecific binding of library DNA to targeted probes. Customized xGen lockdown probe panels (Integrated DNA Technologies) were used for targeted enrichment of 425 predefined genes. The hybridization reaction was performed using NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche). Dynabeads M-270 (Life Technologies) were used to capture probe-bound fragments. Then, the library was amplified with Illumina p5 and p7 primers in KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and purified using Agencourt AMPure XP beads. Library quantification was achieved using the KAPA Library Quantification kit (KAPA Biosystems). The size distribution was measured using Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). Enriched libraries were sequenced on HiSeq 4000 NGS platforms (Illumina) to coverage depths of at least 100 x and 300 x for normal tissue and tumor, respectively, after removing PCR duplicates.

He W., Leng X., Yang Y., Peng L., Shao Y., Li X, & Han Y. (2020). Genetic Heterogeneity of Esophageal Squamous Cell Carcinoma with Inherited Family History. OncoTargets and therapy, 13, 8795-8802.

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Cancer-related gene panel sequencing of cfDNA

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cfDNA or fragmented genomic DNA (300~350bp with Covaris M220 instrument) underwent sequencing library preparation using KAPA Hyper Prep kit (KAPA Biosystems). In brief, DNA was experienced with end-repairing, A-tailing, adapter ligation, size selection using Agencourt AMPure XP beads (Beckman Coulter), and then was amplified by polymerase chain reaction (PCR) and purified before targeted enrichment.
Indexed DNA libraries were pooled up to 2 µg together with Human cot-1 DNA (Life Technologies) and xGen Universal blocking oligos (Integrated DNA Technologies) as blocking reagents. Customized xGen lockdown probes panel (Integrated DNA Technologies) covering 413 predefined cancer-related genes was used to perform hybridization capture. Enriched libraries were sequenced on Hiseq 4000 NGS platforms (Illumina) to targeted mean coverage depths of at least 100x for swab control samples, 500x for tumor or cell genomic DNA and 3000x for cfDNAs.

Zhou L., Zhao H., Shao Y., Chen X., Hong R., Wang L., Ni F., Nagler A., Hu Y, & Huang H. (2021). Serial surveillance by circulating tumor DNA profiling after chimeric antigen receptor T therapy for the guidance of r/r diffuse large B cell lymphoma precise treatment. Journal of Cancer, 12(18), 5423-5431.

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