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当前位置： 首页 > 产品中心 > > NEB/NEBNext® Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1)/384 reactions/E7416L
商品详细NEB/NEBNext® Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1)/384 reactions/E7416L
NEB/NEBNext® Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1)/384 reactions/E7416L
NEB/NEBNext® Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1)/384 reactions/E7416L
商品编号： E7416L
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商品介绍

NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) are built for more streamlined and informative NGS multiplexing, more accurate determination of transcript abundance, and removal of PCR duplicates. These index adaptors contain a pair of unique dual indices (i5 and i7) and one 11 nt UMI sequence. This set includes 96 unique dual index UMI adaptors, packaged in a single-use 96-well plate with a pierceable foil seal. The universal primer mixis supplied in a separate vial.

Combining UMI Adaptors with one of the NEBNext RNA library prep kits is simple and straightforward if you follow the protocols found in the UMI Adaptor-specific library prep kit manuals (listed below).

  • NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760 or NEB #E7765)
  • NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770 or NEB #E7775)
  • NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400 or NEB #E7405)
  • NEBNext rRNA Depletion Kit (Bacteria) (NEB #E7850 or NEB #E7860)
  • NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7750 or NEB #E7755)

The NEBNext line of Multiplex Oligos can be used with NEBNext sample prep products or other standard Illumina-compatible library preparation protocols. In addition to this set, index adaptors with UMIs are available for PCR-free and standard DNA-seq (NEB #E7395). For multiplexing without UMIs, NEBNext offers several options for added flexibility, including unique dual index primers (NEB #6440,NEB #6442, NEB #E6444, NEB #E6446), dual index primers (NEB #E7600andNEB #E7780), and single primer sets (in 12- and 96-index formats; NEB #E7335,NEB #E7500,NEB #E7710,NEB #E7730,NEB #E6609), as well as an option compatible with EM-seq™ and bisulfite sequencing (NEB #E7140).

The NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) workflow shown below details the library barcoding steps of the library preparation process.

Figure 1: NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1) Workflow
Figure 2: NEBNext Unique Dual Index UMI Adaptors have equally efficient ligation and subsequent amplification
A. Universal Human Reference (UHR) RNA (Agilent® #740000) was used to prepare 96 libraries with the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) following NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) enrichment. Library yields were quantified (Agilent Tapestation 4200) and normalized by dividing the individual library yields by the average library yield across all 96 libraries. Adaptor ligation efficiency was robust with uniformity across all 96 Unique Dual Index UMI Adaptors. Each bar represents the average of at least 2 technical replicates.B. NEBNext Unique Dual Index UMI Adaptors have uniform clustering efficiency. 96 libraries were pooled and sequenced on the Illumina NextSeq® 500 (PE70). Each library is expected to contribute 1.04% of the total sequencing reads. Individual library yields were normalized by dividing the number of actual reads by the number of expected reads. No bias was observed across the 96 Unique Dual Index UMI Adaptor libraries.
Figure 3: NEBNext Unique Dual Index UMI Adaptors outperform competitor UMI adaptors
Libraries were produced using UHR RNA and the NEBNext Ultra II Directional RNA library prep kit (NEB #E7760) following NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) enrichment.A. The average library yield of triplicates is shown for three starting total RNA inputs: 10, 100, and 1,000 ng. During adaptor ligation either NEBNext Unique Dual Index UMI Adaptors or IDT xGen dual index adapters were used. Final library yields were quantified using the Agilent Tapestation 4200.B. Libraries were sequenced on the Illumina NextSeq 500 (PE70). Reads were downsampled to 5 million reads (seqtk) and aligned to the GRCh38 reference genome using HISAT 2.1. UMI information was added using fgbio (AnnotateBamWithUmis) and MarkDuplicates (Picard 2.18.2.1) was used to mark reads with UMI and determine duplication rate. NEBNext Unique Dual Index UMI Adaptor libraries produced libraries with a lower percentage of read duplicates.
Figure 4: Incorporating UMIs into analysis improves the accurate quantification of duplication rates
Triplicate libraries of three input amounts (10, 100, and 1000 ng) were produced using UHR RNA and the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) following NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) enrichment and sequenced on the NextSeq 500. Reads were downsampled (seqtk) to 5 million reads and mapped to the GRCh38 reference genome (HISAT 2.1) and Fgbio (AnnotateBamWithUmis) was used to add the UMI information. Two approaches were used to calculate duplication: position and UMI information (green bars) or position alone (yellow bars) using MarkDuplicates (Picard 2.18.2.1).
Figure 5: Unique Dual Index UMI Adaptors allow for the correction of bias introduced with PCR amplification
Libraries were produced using UHR RNA and the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) following NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) enrichment. Four technical replicate libraries were produced at three RNA inputs (10, 100 and 1,000 ng). Libraries were sequenced on the NextSeq 500 (PE70) and downsampled (seqtk)to 10 million reads for further analysis. To assess PCR bias, the same sequencing reads were compared using typical transcript abundance analysis (Salmon 0.9.1) against UMI deduplicated reads. UMI deduplication was performed by first mapping to the GRCh38 reference genome (HISAT 2.1) followed by Fgbio to add the UMI information. Non-duplicate reads were randomly ordered and converted to fastq before re-assessing transcript abundance using Salmon (0.9.1). The ratio of UMI-adjusted counts/raw read counts with no deduplication was calculated. Number of transcripts with at least 1 count in any library and with UMI/raw ratio ≥2 are plotted. In total, approximately 90,000 total transcripts were detected. An increased number of PCR cycles in library preparation correlates with the number of transcripts that shift upon UMI correction.
Figure 6: IGV visual representation of mapped reads adjusted utilizing UMIs in MDM4 (chr1:204,548.089-204,558,681)
Several isoforms of MDM4 locus showed >2X fold change in UMI/raw ratio in a 10 ng library sequenced on the NextSeq 500 (PE70). Reads that are considered to originate from unique molecules are shown in blue. Duplicate reads determined by UMI sequence and mapped read position (red bars) can be removed to improve accuracy of transcript abundance in downstream analysis.

This product is related to the following categories:
NEBNext® Multiplex Oligos (Adaptors & Primers),
Next Generation Sequencing Library Preparation
品牌介绍
New England Biolabs（NEB）公司       NEB公司——三十多年的卓越品质NEB公司成立于二十世纪七十年代中期，拥有众多经验丰富的科学家，是生产生命科学试剂的领导者。目前，NEB为基因组研究提供最齐全的重组酶和天然酶，并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程，NEB公司作为先驱公司之一，为促进生物科技工业的发展做出了巨大的贡献。NEB美国总部乔迁新址后拥有最尖端的设备，有一座现代化的发酵中心及设备齐全的实验室，这些实验室主要用于产品生产、质量监控、产品开发和基础科研之用。作为首批以商业规模生产限制性内切酶的公司之一，NEB一直专注于内切酶的研究，并保持业内领先水平。NEB公司一贯坚持以科学为本的原则，公司生产的试剂因其高质量、高性价比享誉世界。重组酶NEB公司对酶的生产与其基础科研不能分开，通过对限制/修饰系统的克隆和过量表达方面的研究，使我们能够大大降低成本，改善产品质量。NEB已经成功克隆了180多种内切酶，其中大多是完全克隆，少数是部分克隆。目前，NEB可供应240多种内切酶，其中180多种可以重组酶形式提供，同时还有大量的应用途广泛的重组聚合酶和重组修饰酶。质量与客户服务NEB公司凭借着严格的质检程序、深入的基础科研以及不断开发的研发项目，承诺为全球科技人员提供高纯度的科研产品。直接与NEB总部联系或与NEB的国际网点联系，即可体验到NEB的个性化客户服务。公司内负责生产以及负责进行质量监控的科学家（产品负责人）也就是技术支持人员。他们为客户解答有关限制核酸内切酶、甲基化酶、以及其他DNA修饰酶、蛋白质修饰酶方面的问题。同时，公司的有机合成部门可提供linkers、引物、adaptors、探针以及寡核苷酸合成等方面的信息。研究人员也可为客户提供技术支持服务，这些研究人员在DNA测序、甲基化、克隆、过量表达、发酵、蛋白质纯化以及蛋白质分析方面都有很深造诣。基础科研NEB在分子生物学和寄生虫学方面的基础研究由公司内部的资深科学家负责。这些科学家在他们各自的领域都卓有成绩，经常在业内著名杂志上发表文献，指导博士后工作、为大学生提供暑期实习机会，而且经常受邀去当地学校进行演讲。NEB鼓励公司与外界合作，每周学术探讨会为其他科学家们提供了交流和展示的平台。环保政策NEB公司在资助科研项目时最基本原则之一就是：一切工作都应符合保护生态环境。公司产品生产、分析以及运输过程既要满足产品的稳定性，也要符合我们的环保政策。20年来，NEB公司最引人注目的一项环保政策是：回收运输泡沫盒，虽然这项工作首创于美国，但是，目前加拿大、德国以及英国的分公司也同样实施了。公司还在内部开展多方位的回收工作，经常用再生纸张印刷市场宣传资料。这些小细节不会影响产品质量，但是对环境的保护却是显而易见。公司职责除环保政策外，通过基金会，NEB还竭力为改善当地社区及全人类尽微薄之力。NEB基金会创立于1982年，属私人性质，其宗旨是支持发展中国家的环保、教育、健康及艺术事业。此外，NEB捐款委员会也经常向当地社区进行捐款活动。
公司介绍
New England Biolabs（NEB）公司       NEB公司——三十多年的卓越品质NEB公司成立于二十世纪七十年代中期，拥有众多经验丰富的科学家，是生产生命科学试剂的领导者。目前，NEB为基因组研究提供最齐全的重组酶和天然酶，并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程，NEB公司作为先驱公司之一，为促进生物科技工业
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