标签归档：illumina

Next® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3) |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

Incorporating a novel hairpin loop structure, the NEBNext® Adaptor ligates with increased efficiency to end-repaired, dA-tailed DNA. The loop contains a U, which is removed by treatment with USER® Enzyme (a mix of UDG and Endo VIII), to open up the loop and make it available as a substrate for PCR. During PCR, barcodes can be incorporated by use of the NEBNext index primers, thereby enabling multiplexing. This kit includes 96 pre-mixed unique pairs of i5 and i7 index primers, packaged in a single-use 96-well plate with a pierceable foil seal.

NEBNext Oligos can be used with NEBNext products, and with other standard Illumina-compatible library preparation protocols. 

Additional dual barcode options (NEB #E7600, NEB #E7780), and single primer options are also available, in 12- and 96- index formats (NEB #E7335, NEB #E7500, NEB #E7710, NEB #E7730, NEB #E6609). A methylated version of the NEBNext Adaptor is also available for use with bisulfite sequencing protocols (NEB #E7535). 

Workflow:

Designed for use in library prep for DNA, ChIP DNA and RNA (but not Small RNA), the NEBNext Adaptors enable high-efficiency adaptor ligation and high library yields, with minimized adaptor-dimer formation. Incorporating a novel hairpin loop structure, the NEBNext Adaptor ligates with increased efficiency to end-repaired, dA-tailed DNA. The loop contains a U, which is removed by treatment with USER Enzyme (a combination of UDG and Endo VIII), to open up the loop and make it available as a substrate for PCR. During PCR, barcodes can be incorporated by use of the NEBNext index primers, thereby enabling multiplexing. The 96 8-base index primer pairs included in this kit are pre-mixed and are packaged in a single-use 96-well plate with a pierceable foil seal. 

For multiplexing with 384 indices, combine this set with NEB #E6440, NEB #E6442, and NEB #E6446.

Figure 1: NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 3) Workflow

Next® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3) |

Figure 2: Use of NEBNext Adaptor and Unique Dual Index Primer Pairs substantially increases library yields and minimizes adaptor-dimers

Next® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3) |

16 libraries were prepared with 100 ng of Human NA19240 genomic DNA (Coriell Institute), using the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805). Adaptors and primers were from either the IDT® for Illumina –TruSeq® DNA UD Indexes (Illumina #20022370), or the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs), NEB #E6440. After 4 PCR cycles, libraries were quantified on an Agilent® TapeStation® 4000.

A) Average library yields for the 8 libraries made using each workflow show 60% higher yield when the NEBNext Adaptor and Unique Dual Index Primer Pairs are used.

B) TapeStation traces of 16 libraries show the presence of adaptor-dimer (indicated by the orange arrow) with the libraries made using IDT for Illumina adaptors and primers, which is absent in the libraries prepared using NEBNext adaptors and primers, which also have higher yields.

Figure 3: Libraries amplified with NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 3) cluster evenly on the Illumina NovaSeq™ 6000

Next® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3) |

96 libraries produced using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 3) were pooled at equimolar concentrations and sequenced on the Illumina MiSeq® and NovaSeq® 6000 instruments. The total number of reads from all libraries were summed, and the fraction of the total reads contributed by each library was determined (expected fraction per library = 1.04 %). All 96 libraries clustered efficiently and were represented at approximately the expected frequency on both platforms. 

Figure 4: Use of NEBNext Adaptor and Unique Dual Index Primer Pairs substantially increases library yields and minimizes adaptor-dimers

Next® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3) |

Human NA19240 genomic DNA (Coriell Institute) was used to prepare 96 libraries using either the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805), Covaris-sheared DNA with the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645), or the NEBNext Ultra II DNA Library Prep Kit combined with bisulfite conversion. Universal Human Reference RNA was used to prepare 96 libraries using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760). Libraries were PCR-amplified using the NEBNext Multiplex Oligos (96 Unique Dual Index Primer Pairs Set 3) to produce libraries containing unique i5 and i7 indices. Library yields were quantified (Agilent® TapeStation® 4200) and normalized by summing the total yield of all 96 libraries and calculating the contribution from each library (expected fraction per library = 1.04%). Library amplification efficiency was robust with each library prep method, and efficiency was uniform across all 96 unique index primer combinations. Each bar represents the average of at least 2 technical replicates. 

Figure 5: Libraries amplified with 384 primer pairs from NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 1-4) cluster evenly on the Illumina NovaSeq™ 6000

Next® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3) |

384 libraries produced using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 1-4) were pooled at equimolar concentrations and sequenced on the Illumina NovaSeq® 6000 instrument. The total number of reads from all libraries were summed, and the fraction of the total reads contributed by each library was determined (expected fraction per library = 0.26 %). All 384 libraries clustered efficiently and were represented at approximately the expected frequency. 
产品类别:
NEBNext® Multiplex Oligos (Adaptors & Primers) Products,
Next Generation Sequencing Library Preparation Products

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E6444S     -20    
        NEBNext® Adaptor for Illumina® E6612AVIAL -20 1 x 0.96 ml 15 µM
        USER® Enzyme E6610AVIAL -20 1 x 0.288 ml Not Applicable
        NEBNext® Unique Dual Index Primer Set 3 E6445AVIAL -20 1 x 10 μl/well 10 µM
    • E6444L     -20    
        NEBNext® Adaptor for Illumina® E6612AVIAL -20 4 x 0.96 ml 15 µM
        USER® Enzyme E6610AAVIAL -20 2 x 0.576 ml Not Applicable
        NEBNext® Unique Dual Index Primer Set 3 E6445AVIAL -20 4 x 10 μl/well 10 µM

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find protocols for using NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs)? (NEB #E6440, #E6442, #E6444, #E6446, #E6448)

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE6444

  • 使用指南

    • E6444_UDI_IlluminaLRM
    • Forward Strand Workflow Sample Sheet for E6444: NovaSeq 6000 with v1.0 reagent kits, MiniSeq with Rapid reagent kits, MiSeq, HiSeq 2000/2500 (paired-end flow cell), HiSeq 3000/4000 (single-read flow cell)
    • Reverse Complement Workflow Sample Sheet for E6444: iSeq 100, MiniSeq with Standard reagent kits, NextSeq Systems, NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/2500 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell)

工具 & 资源

  • 选择指南

    • NEBNext® Multiplex Oligos Selection Chart

FAQs & 问题解决指南

  • FAQs

    1. Are NEBNext adaptors and primers for Illumina validated in Next Generation Sequencing Workflows?
    2. What is the concentration of the adaptor and primers in the NEBNext Multiplex Oligos kits?
    3. Can I use the 96-well primer plate to do my PCR reactions?
    4. How many reactions worth of primer are in each well?
    5. Is the NEBNext adaptor methylated?
    6. Can I perform single read runs and still get both index sequences? Can I run both single read and paired-end recipes with dual-indexed libraries?
    7. Are dual indexed libraries compatible with single end sequencing?
    8. How many indices are available with NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (NEB #E6440, E6442, E6444, E6446, and E6448)?
    9. Can the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB #E6440, #E6442, #E6444, #E6446, and #E6448) be used to address “index hopping” with Illumina patterned flow cells?
    10. Where can I find the sample sheets for these Multiplex Oligos?
    11. What should I do if the sample sheet from the NEB.com website is not compatible with the software version I am using?
    12. Which sets of NEBNext Unique Dual Index Primer Pairs can be combined in a single Illumina sequencing run?
    13. What sequences need to be trimmed for NEBNext libraries that are sequenced on an Illumina instrument?

Next® 文库定量试剂盒（Illumina®） |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

Next® 文库定量试剂盒（Illumina®） |

图 1：NEBNext 文库定量试剂盒的典型扩增和标准曲线

Next® 文库定量试剂盒（Illumina®） |

图（A）是 NEBNext 文库定量试剂盒在 Bio-Rad CFX96 Touch 上的结果；图（B）是在 Applied Biosystems 7500 快速实时定量 PCR 仪器上的结果。左图显示扩增曲线，右图显示标准曲线。两种仪器都使用了默认设置。ABI 7500 快速分析包含 1X ROX（低浓度）和 ROX 标准品。

NEBNext 文库定量试剂盒优化了针对 Illumina 测序平台基于 qPCR 定量的组份。该试剂盒包含 P5 和 P7 Illumina 接头序列引物，以及一组 6 个高质量预稀释的 DNA 标准品，对 150 – 1,000 bp 之间稀释的 DNA 文库进行可靠的定量。

特性

  • 与其它方法和试剂盒相比，可提供更精准和可重复的定量结果
  • 兼容于通过多种方法制备的具有宽范围插入片段大小及 GC 含量的文库
  • 可根据实验需求灵活选择最佳标准曲线（4 个或 6 个标准品）
  • 提供使用方便的文库稀释缓冲液
  • NEBNext 文库定量预混液只需要加入引物
  • 无论插入片段大小如何，所有文库均使用同一延伸时间
  • 文库定量结果可以通过 NEB 在线工具 NEBioCalculator.neb.com 轻松计算
  • 试剂盒中包含 ROX，方便需要参照染料的 qPCR 仪器进行标准化

每套试剂均通过 qPCR 文库定量分析进行功能验证。试剂通过功能测试，遵守严格检测效率标准及每个 DNA 标准品的 Cq 值和无模板对照反应。

图 2：NEBNext 文库定量试剂盒（Illumina®）工作流程

Next® 文库定量试剂盒（Illumina®） |

图 3：qPCR 法提供比 Bioanalyzer® 分析更一致的文库定量结果

Next® 文库定量试剂盒（Illumina®） |

使用 NEBNext 文库定量试剂盒（Illumina®）（橙色）测定 4 个文库的浓度，并与使用 Agilent Bioanalyzer（蓝色）测定的结果进行比较。与基于 qPCR 的 NEBNext 方法相比，Bioanalyzer 所测定的结果呈现出更大幅度的变化。
图 4：使用 NEBNext 文库定量试剂盒（Illumina®）可显著提高文库定量的可重复性

Next® 文库定量试剂盒（Illumina®） |

4 位用户分别使用 NEBNext 或 KapaTM 文库定量试剂盒（通用），对三个 340 – 400 bp 的文库进行定量，重复 2 – 4 次。与 Kapa 试剂盒（灰色）相比，NEBNext 试剂盒（橙色）对文库浓度定量的一致性有显著改善。
图 5：使用 NEBNext 文库定量试剂盒（Illumina®），标准品的批间差更小、更稳定

Next® 文库定量试剂盒（Illumina®） |

精准的 qPCR 定量需要用已知浓度的高质量的 DNA 标准品。NEBNext 文库定量试剂盒包含 6 个标准品，具有极高的定量准确度和一致性。图中显示了 4 个批次的 NEBNext（橙色）和 Kapa（灰色）标准品 > 70 次运行的总实验数据，并绘制了所有 Cq 值。框和点分别代表平均值和四分位数。NEBNext 文库标准品的 Cq 值变化更小，说明具有更高的文库定量一致性。
图 6：使用 NEBNext 文库定量试剂盒（Illumina®）定量值可获得最佳簇密度

Next® 文库定量试剂盒（Illumina®） |

使用 NEBNext 文库定量试剂盒（橙色）或 Kapa 文库定量试剂盒（通用）（对色）对 7 个不同的文库进行定量。未稀释的文库浓度范围为 2 – 200 nM。将文库稀释至 8 pM 并在 MiSeq® 仪器（v2 化学；MCS v2.4.1.3）上样。使用 NEBNext 试剂盒定量后，文库的原始簇密度平均值为 1,160 k/mm2，在最佳范围 900 – 1,300 k/mm2 内。相比之下，基于 Kapa 定量的文库平均值只有 660 k/mm2。
图 7：使用 NEBNext 文库定量试剂盒（Illumina®），可以从具有广范的文库大小和 GC 含量的量化文库中获得最佳簇密度

Next® 文库定量试剂盒（Illumina®） |

使用 NEBNext 文库定量试剂盒定量来自指定来源的 310–963 bp 的文库，然后稀释至 8 pM 并在 MiSeq 仪器（v2 化学；MCS v2.4.1.3）上样。文库浓度范围为 7 – 120 nM，文库的原始簇密度为 965 – 1,300 k/mm2（ave. = 1,199）。使用 NEBNext 文库定量试剂盒定量的文库大小浓度，都获得最佳簇密度。
图 8：通过广泛的文库大小和 GC 含量实现准确的文库定量
Next® 文库定量试剂盒（Illumina®） |
使用 NEBNext 文库定量试剂盒成功定量一系列文库。按由小到大绘制，小至 150 – 230 bp（sRNA、FFPE），大到 980 bp。使用多种样本来源，GC 含量在 20-70% 之间，图中文字使用不同颜色显示。分别使用 NEBNext、Illumina TruSeq® Nano 和 Kapa Hyper 文库制备试剂盒制备（数据未显示）文库。结果显示，使用 NEBNext 进行文库定量不依赖于大小或 GC 含量。
产品类别:
NGS Library Quantitation Products,
Next Generation Sequencing Library Preparation Products

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E7630S     -20    
        NEBNext® Library Quant Master Mix E7640AVIAL -20 1 x 1.5 ml Not Applicable
        NEBNext® Library Quant Primer Mix E7639AVIAL -20 1 x 0.1 ml Not Applicable
        NEBNext® Library Dilution Buffer E7633AVIAL -20 2 x 1 ml 10 X
        NEBNext® Library Quant DNA Standard 1 E6886AVIAL -20 1 x 0.024 ml 100 pM
        NEBNext® Library Quant DNA Standard 2 E6887AVIAL -20 1 x 0.024 ml 10 pM
        NEBNext® Library Quant DNA Standard 3 E6888AVIAL -20 1 x 0.024 ml 1 pM
        NEBNext® Library Quant DNA Standard 4 E6889AVIAL -20 1 x 0.024 ml 0.1 pM
        NEBNext® Library Quant DNA Standard 5 E6890AVIAL -20 1 x 0.024 ml 0.01 pM
        NEBNext® Library Quant DNA Standard 6 E6891AVIAL -20 1 x 0.024 ml 0.001 pM
        ROX (High) E7641AVIAL -20 1 x 0.02 ml Not Applicable
        ROX (Low) E7638AVIAL -20 1 x 0.02 ml Not Applicable
    • E7630L     -20    
        NEBNext® Library Quant Master Mix E7640AAVIAL -20 1 x 7.5 ml Not Applicable
        NEBNext® Library Quant Primer Mix E7639AAVIAL -20 1 x 0.5 ml Not Applicable
        NEBNext® Library Dilution Buffer E7633AAVIAL -20 2 x 7.5 ml 10 X
        NEBNext® Library Quant DNA Standard 1 E6886AAVIAL -20 1 x 0.12 ml 100 pM
        NEBNext® Library Quant DNA Standard 2 E6887AAVIAL -20 1 x 0.12 ml 10 pM
        NEBNext® Library Quant DNA Standard 3 E6888AAVIAL -20 1 x 0.12 ml 1 pM
        NEBNext® Library Quant DNA Standard 4 E6889AAVIAL -20 1 x 0.12 ml 0.1 pM
        NEBNext® Library Quant DNA Standard 5 E6890AAVIAL -20 1 x 0.12 ml 0.01 pM
        NEBNext® Library Quant DNA Standard 6 E6891AAVIAL -20 1 x 0.12 ml 0.001 pM
        ROX (High) E7641AAVIAL -20 1 x 0.1 ml Not Applicable
        ROX (Low) E7638AAVIAL -20 1 x 0.1 ml Not Applicable

  • 特性和用法

    需要但不提供的材料

    • 无核酶水
    • qPCR 仪器
    • qPCR 板和密封件
    • PCR 联管或微型离心管
    • 锥形离心管

  • 优势和特性

    Features

    • 与其它方法和试剂盒相比，可提供更精准和可重复的定量结果
    • 兼容于通过多种方法制备的具有宽范围插入片段大小及 GC 含量的文库
    • 可根据实验需求灵活选择最佳标准曲线（4 个或 6 个标准品）
    • 提供使用方便的文库稀释缓冲液
    • NEBNext 文库定量预混液只需要加入引物
    • 无论插入片段大小如何，所有文库均使用同一延伸时间
    • 文库定量结果可以通过 NEB 在线工具 NEBioCalculator.neb.com 轻松计算
    • 试剂盒中包含 ROX，方便需要参照染料的 qPCR 仪器进行标准化

  • 相关产品

    相关产品

    • NEBNext® 文库稀释缓冲液
    • E7642 NEBNext 文库定量 DNA 标准品

  • 注意事项

    1. 试剂盒组份的贮存方法：
      NEBNext 文库定量试剂盒（Illumina）的所有单独组份均可在 –20℃ 条件下稳定贮存两年。未使用时，试剂盒组份应于 –20℃ 条件下贮存。该试剂盒的所有组份（包括 NEBNext 文库定量预混液和引物混合液）至少经历 30 次反复冻融都能保持稳定。

      一旦将引物混合液添加到 NEBNext 文库定量预混液中后（请参阅手册第 6 页的“操作说明”），所得混合液可在 –20℃ 条件下贮存七个月。如需短期贮存，该混合液可在 4℃ 条件下贮存最多六周。

      可在室温条件下配制反应，但试剂盒中未使用的试剂在解冻后应放在冰上使用。

    2. 对于提供的试剂：

      除试剂盒中提供的稀释缓冲液以外，如果您还需要稀释缓冲液，可以单独购买。

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find protocols for using the NEBNext Library Quant Kit?

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE7630_E7642

  • 应用实例

    • Improved library quantitation for a broad range of library types using the NEBNext® Library Quant Kit for Illumina®

FAQs & 问题解决指南

  • FAQs

    1. How are the new 6 standards different from the old 4 standards?
    2. What are the advantages of the qPCR-based NEBNext Library Quant Kit compared to other methods of quantitating NGS libraries?
    3. What libraries can be quantitated with the Quant Kit?
    4. How much should I dilute my library to run in the Quant Kit?
    5. Can I run the NEBNext Library Quant Kit on my qPCR machine?
    6. Which ROX should I use for my qPCR machine?
    7. Are 4 standards enough for a qPCR quantitation?
    8. Does the library quant require a size correction?
    9. What other materials/information do I need to run the kit?
    10. How many libraries can I quantitate at a time?
    11. How long is a run of the Quant Kit?
    12. Do you provide any analysis tools to help with the quantitation?
    13. Can I see adaptor dimers as a measure of library quality?

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2) |NEB酶试剂 New England Biolabs

发表于由

上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 2) are built for more streamlined and informative NGS multiplexing, tracking the sequencing reaction at the individual molecule level and enabling bioinformatic removal of misassigned reads for optimal accuracy. These index adaptors contain a pair of unique dual indices (i5 and i7) and one UMI sequence, making the resulting library multiplex-ready, with or without PCR. 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 mix is supplied in a separate vial.

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, there are three other sets of NEBNext Unique Dual Index UMI Adaptors for DNA (NEB #E7395, #E7876, #E7878) for multiplexing of up to 384 samples; additional index adaptors with UMIs are available for RNA-seq (NEB #E7416). For multiplexing without UMIs, NEBNext offers several options for added flexibility, including unique dual index primers (NEB #6440, NEB #6442, NEB #E6444, NEB #E6446, and NEB #E6448), dual index primers (NEB #E7600 and NEB #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). 

For larger volume requirements,  customized and/or bulk packaging is available through our Customized Solutions Team. Please contact us at [email protected].

NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 2) enable both PCR-free and amplification-based workflows for DNA library preparation. Refer to the below diagram for the differences between the two options.

Figure 1. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 2) Workflow

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2) |

Figure 2. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 2) allow higher ligation efficiency.

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2) |

A. Libraries were prepared with 100, 250, and 500 ng inputs of human cell line NA19240 genomic DNA (Coriell Institute for Biomedical Research) and unique dual index adaptors from different suppliers using the NEBNext Ultra™ II FS DNA Library Prep Kit (NEB #E7805) without PCR amplification. The adaptors used were NEBNext Unique Dual Index UMI Adaptrs, xGen® Dual Index UMI Adapters (IDT®) and Nextflex® Unique Dual index Barcodes 1-96 (Bioo Scientific®). Two rounds of SPRIselect bead-based cleanup steps were performed post-ligation, and libraries were quantified by qPCR (NEBNext Library Quant Kit, NEB #E7630).  
B. 90 libraries were prepared with 100 ng NA19240 genomic DNA using the Ultra II FS DNA Library Prep Kit without PCR amplification in a 96-well plate. For the 90 libraries, 30 different adaptors each from NEB, IDT, and Bioo were used for ligation. Libraries were cleaned up and quantitated by qPCR. 
Figure 3. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 2) allow more-sensitive, low-frequency variant detection.

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2) |

AcroMetrix® Oncology Hotspot Control DNA (Thermo Scientific®) was used as a mutation DNA source (> 500 mutations with 5-35% allele frequency) and mixed with NA19240 genomic DNA at various ratios to generate a range of allele frequencies (5%-35%, 2.5%-17.5% and 1%-7%). Libraries were constructed using NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) with NEBNext Unique Dual Index UMI Adaptors, and multiplex hybrid capture was performed on all samples using a customized panel for 152 genes from Twist Bioscience®. Libraries were sequenced on a NovaSeq® 6000 (PE140), and reads were downsampled to 110 Million (Seqtk Sample) and mapped to GRCh38 with BWA MEM (0.7.17). Mapped reads were analyzed by MarkDuplicates (Picard 2.20.6) without utilizing UMI information or by building UMI consensus sequences (Fgbio 0.8.1). The final BAM files were used to call somatic variants with Strelka2 (2.9.10).
A. The number of total and correct SNV calls increased when using UMI information for duplicate removal and consensus sequence-based error correction. 
B. The sensitivity of variant detection was improved with UMI consensus calling. The lower the allele frequency, the more benefit provided by UMI information in SNV detection.
Figure 4. NEBNext Unique Dual Index UMI Adaptors DNA Set 2 provides consistent library yields

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2) |

96 Libraries were each prepared with 50 ng input of human cell line NA19240 genomic DNA (Coriell Institute for Biomedical Research) and the NEBNext Unique Dual Index UMI Adaptors DNA Set 2 using the NEBNext Ultra II FS PCR-free DNA Library Prep Kit (NEB #E6177) in a 96 well plate. Library cleanup was followed by qPCR quantification. Library concentrations ranged from 7.3 nM to 3.4 nM with a 2.1-fold difference across all 96 adaptors.
Figure 5.  NEBNext Unique Dual Index UMI Adaptors DNA Set 2 provides consistent clustering efficiency on MiSeq® and NovaSeq 6000

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2) | 

Fig Desc: 96 libraries prepared using the NEBnext Unique Dual Index UMI Adaptors DNA Set 2 and the NEBNext Ultra II FS PCR-free DNA Library Prep Kit (NEB #E6177) were pooled at equimolar concentrations and sequenced on the Illumina MiSeq and NovaSeq 6000 instruments. The total number of reads from all libraries were summed, and the fraction of the total reads contributed by each library was determined (expected faction per library = 1.04%). All 96 libraries clustered efficiently and were represented at approximately the expected frequency on both platforms.
Figure 6.  All libraries prepared with 384 NEBNext Unique Dual Index UMI Adaptors cluster evenly on the Illumina NovaSeq 6000

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2) | 

384 libraries prepared using the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 and the NEBNext Ultra II FS PCR-free Library Prep Kit (NEB #E6177) were pooled at equimolar concentrations and sequenced on the Illumina NovaSeq 6000 instrument. The total number of reads from all libraries was summed, and the fraction of the total reads contributed by each library was determined (expected fraction per library = 0.26%). All 384 libraries clustered efficiently and were represented at approximately the expected frequency.
产品类别:
NEBNext® Multiplex Oligos (Adaptors & Primers) Products,
Next Generation Sequencing Library Preparation Products

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E7874S     -20    
        NEBNext® UMI DNA Adaptor Plate Set 2 E7875AVIAL -20 1 x 2.5 μl/well 20 µM
        NEBNext® Primer Mix E7397AVIAL -20 1 x 0.48 ml Not Applicable
        NEBNext® UMI Adaptor Dilution Buffer E7398AVIAL -20 2 x 2.5 ml Not Applicable
    • E7874L     -20    
        NEBNext® UMI DNA Adaptor Plate Set 2 E7875AVIAL -20 4 x 2.5 μl/well 20 µM
        NEBNext® Primer Mix E7397AAVIAL -20 2 x 0.96 ml Not Applicable
        NEBNext® UMI Adaptor Dilution Buffer E7398AAVIAL -20 1 x 20 ml Not Applicable

  • 相关产品

    相关产品

    • NEBNext® 多样本接头引物试剂盒 1（Unique 双端 Index 引物，UMI 接头，适用于 DNA）
    • e7876nebnext-multiplex-oligos-for-illumina-unique-dual-index-umi-adaptors-dna-set-3
    • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 4)

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find protocols for using NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 (NEB #E7395, #E7874, #E7876, #E7878) with NEBNext Ultra II reagents?

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE7395_E7874_E7876_E7878
    • manualE6177_E7805 w UMI Adaptors DNA
    • manualE7103_E7645 w UMI Adaptors DNA
    • manualE7595 w UMI Adaptors DNA

  • 使用指南

    • Forward Strand Workflow Sample Sheet for E7874 UMI: NovaSeq 6000 with v1.0 reagent kits, MiniSeq with Rapid reagent kits, MiSeq, HiSeq 2000/2500 (paired-end flow cell), HiSeq 3000/4000 (single-read flow cell)
    • Forward Strand Workflow Sample Sheet for E7874: NovaSeq 6000 with v1.0 reagent kits, MiniSeq with Rapid reagent kits, MiSeq, HiSeq 2000/2500 (paired-end flow cell), HiSeq 3000/4000 (single-read flow cell)
    • Reverse Complement Workflow Sample Sheet for E7874 UMI: iSeq 100, MiniSeq with Standard reagent kits, NextSeq Systems, NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/2500 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell)
    • Reverse Complement Workflow Sample Sheet for E7874: iSeq 100, MiniSeq with Standard reagent kits, NextSeq Systems, NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/2500 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell)

工具 & 资源

  • 选择指南

    • NEBNext® Multiplex Oligos Selection Chart

FAQs & 问题解决指南

  • FAQs

    1. What is the difference between NEBNext Unique Dual Index UMI Adaptors and NEBNext Adaptors for use with multiplex primers?
    2. What are the main applications for the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4?
    3. How long are the UMIs and where are they on the adaptors?
    4. How are UMIs sequenced, and where can I find them?
    5. Can I skip UMI sequencing if I don’t need the UMIs for my applications?
    6. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 compatible with NEBNext reagents for Illumina library preparation?
    7. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 compatible with commercially available library prep reagents?
    8. How many reactions worth of the UMI adaptors are in each well?
    9. Are the adaptors methylated?
    10. What is the concentration of the adaptors and PCR primer mix in the NEBNext Multiplex Oligos (Unique Dual Index UMI Adaptors DNA Sets 1-4)?
    11. How many indices are available with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Sets1-4)? 
    12. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 validated in next generation sequencing workflows?
    13. Are the NEBNext UMI Adaptors compatible with single-end and paired-end sequencing?
    14. Do I need to spike in custom sequencing primers when sequencing libraries made with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Sets 1-4) on the Illumina platforms? 
    15. Can I perform single read runs and still get both index sequences? Can I run both single read and paired-end recipes with dual-indexed libraries?
    16. Where can I find the sample sheets for the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4?
    17. What sequences need to be trimmed for NEBNext libraries that are sequenced on an Illumina instrument?

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3) |NEB酶试剂 New England Biolabs

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产品信息

NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) are built for more streamlined and informative NGS multiplexing, tracking the sequencing reaction at the individual molecule level and enabling bioinformatic removal of misassigned reads for optimal accuracy. These index adaptors contain a pair of unique dual indices (i5 and i7) and one UMI sequence, making the resulting library multiplex-ready, with or without PCR. 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 mix is supplied in a separate vial.

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, there are three other sets of NEBNext Unique Dual Index UMI Adaptors for DNA (NEB #E7395, #E7874, #E7878) for multiplexing of up to 384 samples; additional index adaptors with UMIs are available for RNA-seq (NEB #E7416). For multiplexing without UMIs, NEBNext offers several options for added flexibility, including unique dual index primers (NEB #6440, NEB #6442, NEB #E6444, NEB #E6446, and NEB #E6448), dual index primers (NEB #E7600 and NEB #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). 

For larger volume requirements,  customized and/or bulk packaging is available through our Customized Solutions Team. Please contact us at [email protected].

NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) enable both PCR-free and amplification-based workflows for DNA library preparation. Refer to the below diagram for the differences between the two options.

Figure 1. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) Workflow

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3) |

Figure 2. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) allow higher ligation efficiency.

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3) |

A. Libraries were prepared with 100, 250, and 500 ng inputs of human cell line NA19240 genomic DNA (Coriell Institute for Biomedical Research) and unique dual index adaptors from different suppliers using the NEBNext Ultra™ II FS DNA Library Prep Kit (NEB #E7805) without PCR amplification. The adaptors used were NEBNext Unique Dual Index UMI Adaptrs, xGen® Dual Index UMI Adapters (IDT®) and Nextflex® Unique Dual index Barcodes 1-96 (Bioo Scientific®). Two rounds of SPRIselect bead-based cleanup steps were performed post-ligation, and libraries were quantified by qPCR (NEBNext Library Quant Kit, NEB #E7630).  
B. 90 libraries were prepared with 100 ng NA19240 genomic DNA using the Ultra II FS DNA Library Prep Kit without PCR amplification in a 96-well plate. For the 90 libraries, 30 different adaptors each from NEB, IDT, and Bioo were used for ligation. Libraries were cleaned up and quantitated by qPCR. 
Figure 3. NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 3) allow more-sensitive, low-frequency variant detection.

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3) |

AcroMetrix® Oncology Hotspot Control DNA (Thermo Scientific®) was used as a mutation DNA source (> 500 mutations with 5-35% allele frequency) and mixed with NA19240 genomic DNA at various ratios to generate a range of allele frequencies (5%-35%, 2.5%-17.5% and 1%-7%). Libraries were constructed using NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) with NEBNext Unique Dual Index UMI Adaptors, and multiplex hybrid capture was performed on all samples using a customized panel for 152 genes from Twist Bioscience®. Libraries were sequenced on a NovaSeq® 6000 (PE140), and reads were downsampled to 110 Million (Seqtk Sample) and mapped to GRCh38 with BWA MEM (0.7.17). Mapped reads were analyzed by MarkDuplicates (Picard 2.20.6) without utilizing UMI information or by building UMI consensus sequences (Fgbio 0.8.1). The final BAM files were used to call somatic variants with Strelka2 (2.9.10).
A. The number of total and correct SNV calls increased when using UMI information for duplicate removal and consensus sequence-based error correction. 
B. The sensitivity of variant detection was improved with UMI consensus calling. The lower the allele frequency, the more benefit provided by UMI information in SNV detection.
Figure 4. NEBNext Unique Dual Index UMI Adaptors DNA Set 3 provides consistent library yields

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3) |

96 Libraries were each prepared with 50 ng input of human cell line NA19240 genomic DNA (Coriell Institute for Biomedical Research) and the NEBNext Unique Dual Index UMI Adaptors DNA Set 3 using the NEBNext Ultra II FS PCR-free DNA Library Prep Kit (NEB #E6177) in a 96 well plate. Library cleanup was followed by qPCR quantification. Library concentrations ranged from 7.3 nM to 3.4 nM with a 2.1-fold difference across all 96 adaptors.
Figure 5.  NEBNext Unique Dual Index UMI Adaptors DNA Set 3 provides consistent clustering efficiency on MiSeq® and NovaSeq 6000

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3) |

Fig Desc: 96 libraries prepared using the NEBnext Unique Dual Index UMI Adaptors DNA Set 2 and the NEBNext Ultra II FS PCR-free DNA Library Prep Kit (NEB #E6177) were pooled at equimolar concentrations and sequenced on the Illumina MiSeq and NovaSeq 6000 instruments. The total number of reads from all libraries were summed, and the fraction of the total reads contributed by each library was determined (expected faction per library = 1.04%). All 96 libraries clustered efficiently and were represented at approximately the expected frequency on both platforms.
Figure 6.  All libraries prepared with 384 NEBNext Unique Dual Index UMI Adaptors cluster evenly on the Illumina NovaSeq 6000

Next® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3) | 

384 libraries prepared using the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 and the NEBNext Ultra II FS PCR-free Library Prep Kit (NEB #E6177) were pooled at equimolar concentrations and sequenced on the Illumina NovaSeq 6000 instrument. The total number of reads from all libraries was summed, and the fraction of the total reads contributed by each library was determined (expected fraction per library = 0.26%). All 384 libraries clustered efficiently and were represented at approximately the expected frequency.
产品类别:
NEBNext® Multiplex Oligos (Adaptors & Primers) Products,
Next Generation Sequencing Library Preparation Products

  • 试剂盒组成

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • E7876S     -20    
        NEBNext® UMI DNA Adaptor Plate Set 3 E7877AVIAL -20 1 x 2.5 μl/well 20 µM
        NEBNext® Primer Mix E7397AVIAL -20 1 x 0.48 ml Not Applicable
        NEBNext® UMI Adaptor Dilution Buffer E7398AVIAL -20 2 x 2.5 ml Not Applicable
    • E7876L     -20    
        NEBNext® UMI DNA Adaptor Plate Set 3 E7877AVIAL -20 4 x 2.5 μl/well 20 µM
        NEBNext® Primer Mix E7397AAVIAL -20 2 x 0.96 ml Not Applicable
        NEBNext® UMI Adaptor Dilution Buffer E7398AAVIAL -20 1 x 20 ml Not Applicable

  • 相关产品

    相关产品

    • NEBNext® 多样本接头引物试剂盒 1（Unique 双端 Index 引物，UMI 接头，适用于 DNA）
    • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2)
    • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 4)

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find protocols for using NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 (NEB #E7395, #E7874, #E7876, #E7878) with NEBNext Ultra II reagents?

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualE7395_E7874_E7876_E7878
    • manualE6177_E7805 w UMI Adaptors DNA
    • manualE7103_E7645 w UMI Adaptors DNA
    • manualE7595 w UMI Adaptors DNA

  • 使用指南

    • Forward Strand Workflow Sample Sheet for E7876 UMI: NovaSeq 6000 with v1.0 reagent kits, MiniSeq with Rapid reagent kits, MiSeq, HiSeq 2000/2500 (paired-end flow cell), HiSeq 3000/4000 (single-read flow cell)
    • Forward Strand Workflow Sample Sheet for E7876: NovaSeq 6000 with v1.0 reagent kits, MiniSeq with Rapid reagent kits, MiSeq, HiSeq 2000/2500 (paired-end flow cell), HiSeq 3000/4000 (single-read flow cell)
    • Reverse Complement Workflow Sample Sheet for E7876 UMI: iSeq 100, MiniSeq with Standard reagent kits, NextSeq Systems, NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/2500 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell)
    • Reverse Complement Workflow Sample Sheet for E7876: iSeq 100, MiniSeq with Standard reagent kits, NextSeq Systems, NovaSeq 6000 with v1.5 reagent kits, HiSeq 2000/2500 (single-read flow cell), HiSeq 3000/4000 (paired-end flow cell)

工具 & 资源

  • 选择指南

    • NEBNext® Multiplex Oligos Selection Chart

FAQs & 问题解决指南

  • FAQs

    1. What is the difference between NEBNext Unique Dual Index UMI Adaptors and NEBNext Adaptors for use with multiplex primers?
    2. What are the main applications for the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4?
    3. How long are the UMIs and where are they on the adaptors?
    4. How are UMIs sequenced, and where can I find them?
    5. Can I skip UMI sequencing if I don’t need the UMIs for my applications?
    6. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 compatible with NEBNext reagents for Illumina library preparation?
    7. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 compatible with commercially available library prep reagents?
    8. How many reactions worth of the UMI adaptors are in each well?
    9. Are the adaptors methylated?
    10. What is the concentration of the adaptors and PCR primer mix in the NEBNext Multiplex Oligos (Unique Dual Index UMI Adaptors DNA Sets 1-4)?
    11. How many indices are available with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Sets1-4)? 
    12. Are the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4 validated in next generation sequencing workflows?
    13. Are the NEBNext UMI Adaptors compatible with single-end and paired-end sequencing?
    14. Do I need to spike in custom sequencing primers when sequencing libraries made with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Sets 1-4) on the Illumina platforms? 
    15. Can I perform single read runs and still get both index sequences? Can I run both single read and paired-end recipes with dual-indexed libraries?
    16. Where can I find the sample sheets for the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4?
    17. What sequences need to be trimmed for NEBNext libraries that are sequenced on an Illumina instrument?