Next® 酶学转化法甲基化建库试剂盒 |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

在我们的技术说明中查看或下载更多的性能数据。

NEBNext 酶学转化法甲基化建库试剂盒提供了一种高性能的酶学方法，能够代替重亚硫酸盐转化，通过 Illumina® 平台测序来研究 DNA 甲基化。

DNA 起始量低至 10 ng，使用随附的 NEBNext Ultra II 试剂和经过优化的 EM-seq 接头来制备文库。随后，由 TET2 氧化 5-mC 和 5-hmC 提供保护，避免在后续步骤中被 APOBEC 脱氨基。相反，未修饰的胞嘧啶被脱氨基成尿嘧啶。随后使用 NEBNext Q5U®预混液（Q5® 超保真 DNA 聚合酶的改良版本）扩增文库，再使用 Illumina 仪器进行测序。

高效稳定的 EM-Seq 方法可最大程度地减少对 DNA 的损伤，再结合高效的 Ultra II 文库制备试剂盒，可从有限的测序 read 中更灵敏地检测 CpG 位点。

特性：

  • 更卓越的 5-mC 和 5-hmC 检测灵敏度
  • 更高的比对率
  • 更均一的 GC 覆盖度
  • 可从有限的测序 read 检测到更多的 CpG 位点
  • 更均一的二核苷酸分布
  • 更高效的建库流程，更长的文库插入片段
  • 转化模块可单独购买
图 1：NEBNext 酶学转化法甲基化建库和重亚硫酸盐转化方法

Next® 酶学转化法甲基化建库试剂盒 |

图 2：相较于 WGBS，NEBNext Enzymatic Methyl-seq 能在更低的测序覆盖深度下检测到更多的 CpG 位点

A. Next® 酶学转化法甲基化建库试剂盒 |

B. Next® 酶学转化法甲基化建库试剂盒 |
采用 Covaris® S2 仪器，将 10 ng、50 ng 和 200 ng 不同起始量的人 NA12878 基因组 DNA 打断至 300 bp，同时作为 EM-Seq 和 WGBS 建库流程的起始样本。对于 WGBS 方法，使用 NEBNext Ultra II DNA 进行建库，随后采用 Zymo Research EZ DNA Methylation-Gold™ 试剂盒进行重亚硫酸盐转化。两个文库均使用 Illumina NovaSeq® 6000（2 X 100 base）测序。再使用 bwa-meth 0.2.2 将 read 与 hg38 进行比对。通过分析 3.24 亿个双端 read 得到 EM-Seq 和 WGBS 文库的 CpG 位点覆盖度。

A：其中每条正链和负链均独立计数，最终得到最多 5,600 万个可能的 CpG 位点。结果显示，EM-Seq 能在更低的测序深度检测到更多的 CpG 位点。

B：图中显示了 EM-Seq 和 WGBS 两种方法在不同起始量，至少 1X 和 8X 测序深度下检测到的独有和共有的 CpG 位点。EM-Seq 在至少 1X 测序深度下比 WGBS 多检测到 20% 以上的 CpG 位点。而在至少 8X 测序深度下，CpG 位点覆盖度差异增加至 2 倍。

在我们的技术说明中查看更多性能数据。
图 3：NEBNext Enzymatic Methyl-seq 能实现更卓越的 GC 均一覆盖度

Next® 酶学转化法甲基化建库试剂盒 |

采用 Covaris S2 仪器，将 10 ng、50 ng 和 200 ng 不同起始量的人 NA12878 基因组 DNA 打断至 300 bp，同时作为 EM-Seq 和 WGBS 建库流程的起始样本。对于 WGBS 方法，使用 NEBNext Ultra II DNA 进行建库，随后采用 Zymo Research EZ DNA Methylation-Gold 试剂盒进行重亚硫酸盐转化。两个文库均使用 Illumina NovaSeq 6000（2 X 100 base）测序。再使用 bwa-meth 0.2.2 将 read 与 hg38 进行比对。使用 Picard 2.17.2 计算 GC 覆盖度，图中显示不同 GC 含量时（0-100%），标准化后覆盖度的分布情况。结果显示，EM-Seq 文库显著提高了 GC 覆盖度的均一性，无 AT 过度测序，也无 GC 测序不足：后两项都是 WGBS 文库的典型缺陷。
在我们的技术说明中查看更多性能数据。
图 4：NEBNext 酶学转化法甲基化文库可获得更长的插入片段

Next® 酶学转化法甲基化建库试剂盒 |

采用 Covaris S2 仪器，将 50 ng 人 NA12878 基因组 DNA 打断至 300 bp，同时作为 EM-Seq 和 WGBS 建库流程的起始样本。对于 WGBS 方法，使用 NEBNext Ultra II DNA 进行建库，随后采用 Zymo Research EZ DNA Methylation-Gold 试剂盒进行重亚硫酸盐转化。两个文库均使用 Illumina MiSeq®（2 x 76 base）测序，片段大小采用 Picard 2.18.14 测定。图中绘制了每个插入片段出现的频率标准化后的数据，表明：EM-Seq 建库后的插入片段比 WGBS 方法得到的插入片段更长；说明：EM-seq 不会对 DNA 造成损伤，而 WGBS 方法中重亚硫酸盐处理的 DNA 有严重损伤。
在我们的技术说明中查看更多性能数据。
 
 
产品类别:
Automation for NEBNext® NGS Library Prep Products,
Methylome Analysis Products,
Epigenetic Analysis Products,

Epigenetics Products

应用:
DNA Methylation Analysis,
Bisulfite Conversion,
Methylated DNA Analysis Products ,

Epigenetics

  • 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
    • E7120S     Multi-temperature    
        NEBNext® Enzymatic Methyl-seq Kit E7120-2 -20    
        Control DNA CpG methylated pUC19 E7122AVIAL -20 1 x 0.024 ml 0.1 ng/µl
        Control DNA CpG unmethylated Lambda E7123AVIAL -20 1 x 0.024 ml 2 ng/µl
        NEBNext® Ultra II End Prep Reaction Buffer E7647AVIAL -20 1 x 0.168 ml Not Applicable
        NEBNext® Ultra II End Prep Enzyme Mix E7646AVIAL -20 1 x 0.072 ml Not Applicable
        NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml Not Applicable
        NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml Not Applicable
        Elution Buffer E7124AVIAL -20 1 x 2.1 ml Not Applicable
        TET2 Reaction Buffer E7126AVIAL -20 1 x 0.3 ml Not Applicable
        TET2 Reaction Buffer Supplement E7127AVIAL -20 3 x 100 µl 100 X
        Oxidation Supplement E7128AVIAL -20 1 x 0.024 ml Not Applicable
        Oxidation Enhancer E7129AVIAL -20 1 x 0.024 ml Not Applicable
        TET2 E7130AVIAL -20 1 x 0.096 ml Not Applicable
        Fe (II) Solution E7131AVIAL -20 1 x 0.024 ml Not Applicable
        Stop Reagent E7132AVIAL -20 1 x 0.024 ml Not Applicable
        APOBEC E7133AVIAL -20 1 x 0.024 ml Not Applicable
        APOBEC Reaction Buffer E7134AVIAL -20 1 x 0.24 ml Not Applicable
        BSA E7135AVIAL -20 1 x 0.024 ml Not Applicable
        NEBNext® Q5U®  Master Mix E7136AVIAL -20 1 x 0.6 ml Not Applicable
        DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM
        NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) E7140S -20    
        EM-seq™ Index Primer 1 (S) E7141AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 2 (S) E7142AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 3 (S) E7143AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 4 (S) E7144AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 5 (S) E7145AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 6 (S) E7146AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 7 (S) E7147AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 8 (S) E7148AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 9 (S) E7149AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 10 (S) E7150AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 11 (S) E7151AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 12 (S) E7152AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 13 (S) E7153AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 14 (S) E7154AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 15 (S) E7155AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 16 (S) E7156AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 17 (S) E7157AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 18 (S) E7158AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 19 (S) E7159AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 20 (S) E7160AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 21 (S) E7161AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 22 (S) E7162AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 23 (S) E7163AVIAL -20 1 x 0.005 ml 10 µM
        EM-seq™ Index Primer 24 (S) E7164AVIAL -20 1 x 0.005 ml 10 µM
        NEBNext® EM-seq™ Adaptor E7165AVIAL -20 1 x 0.06 ml Not Applicable
        NEBNext® Sample Purification Beads E7137S 25    
        NEBNext® Sample Purification Beads E7137AVIAL 25 1 x 8.6 ml Not Applicable
    • E7120L     Multi-temperature    
        NEBNext® Enzymatic Methyl-seq Kit E7120-3 -20    
        Control DNA CpG methylated pUC19 E7122AAVIAL -20 1 x 0.096 ml 0.1 ng/µl
        Control DNA CpG unmethylated Lambda E7123AAVIAL -20 1 x 0.096 ml 2 ng/µl
        NEBNext® Ultra II End Prep Reaction Buffer E7647AAVIAL -20 1 x 0.672 ml Not Applicable
        NEBNext® Ultra II End Prep Enzyme Mix E7646AAVIAL -20 1 x 0.288 ml Not Applicable
        NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml Not Applicable
        NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml Not Applicable
        Elution Buffer E7124AAVIAL -20 1 x 8.6 ml Not Applicable
        TET2 Reaction Buffer E7126AAVIAL -20 1 x 1.2 ml Not Applicable
        TET2 Reaction Buffer Supplement E7127AAVIAL -20 3 x 400 µl 100 X
        Oxidation Supplement E7128AAVIAL -20 1 x 0.096 ml Not Applicable
        Oxidation Enhancer E7129AAVIAL -20 1 x 0.096 ml Not Applicable
        TET2 E7130AAVIAL -20 1 x 0.384 ml Not Applicable
        Fe (II) Solution E7131AAVIAL -20 1 x 0.096 ml Not Applicable
        Stop Reagent E7132AAVIAL -20 1 x 0.096 ml Not Applicable
        APOBEC E7133AAVIAL -20 1 x 0.096 ml Not Applicable
        APOBEC Reaction Buffer E7134AAVIAL -20 1 x 0.96 ml Not Applicable
        BSA E7135AAVIAL -20 1 x 0.096 ml Not Applicable
        NEBNext® Q5U®  Master Mix E7136AAVIAL -20 2 x 1.2 ml Not Applicable
        DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM
        NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) E7140L -20    
        NEBNext® 96 Unique Dual Index Primer Pairs Plate (L) E7166AVIAL -20 1 x 5 μl/well 10 µM
        NEBNext® EM-seq™ Adaptor E7165AAVIAL -20 1 x 0.24 ml Not Applicable
        NEBNext® Sample Purification Beads E7137L 25    
        NEBNext® Sample Purification Beads E7137AAVIAL 25 1 x 34.6 ml Not Applicable

  • 特性和用法

    需要但不提供的材料

    • Covaris® S2 仪器或其它打断仪
    • PCR 联管
    • 甲酰胺（Sigma #F9037-100 ml）或 0.1 N NaOH
    • 80% 乙醇 • 0.1X TE，pH 8.0
    • 无核酶水
    • 磁力架/支架，例如 NEBNext 磁性分离架（NEB #S1515）
    • PCR 仪
    • Bioanalyzer®、TapeStation® 和相关耗材或其它片段化分析仪

  • 注意事项

    1. FE(II) 溶液可能呈黄色。检测证明，颜色与性能无直接关系。

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find protocols for using the NEBNext® Enzymatic Methyl-seq Kit? (NEB #E7120, E7125, E7140)

  • 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。

    • manualE7120

  • 使用指南

    • E7140L_E7120L_EMSeqUDI_IlluminaLRM
    • E7140S_E7120S_EMSeqUDI_IlluminaLRM
    • Forward Strand Workflow Sample Sheet for E7140L: 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 E7140S: 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 E7140L: 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 E7140S: 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)

FAQs & 问题解决指南

  • FAQs

    1. What types of sample can be processed using the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
    2. What are the recommended inputs for the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
    3. What is the difference between the NEBNext Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module?
    4. What buffers are recommended for shearing DNA in NEB’s enzymatic methyl-seq (EM-seq™) workflows?
    5. What is the concentration of the EM-seq Adaptor and EM-seq Index Primers?
    6. Can the “active” TET2 Buffer be stored longer than 4 months?
    7. Can the freshly diluted Fe(II) Solution be stored long term?
    8. Why, at some stages of the EM-seq protocol, do the NEBNext Sample Purification Beads behave differently when cleaning up the sample?
    9. What is the expected size of an EM-seq library?
    10. How should EM-seq libraries be sequenced?
    11. How should EM-seq sequencing data be analyzed?
    12. Can the EM-seq Adaptor be substituted with another adaptor?
    13. Are EM-seq libraries directional or non-directional?
    14. Can other buffers be used in place of the supplied EM-seq Elution Buffer?
    15. How are EM-seq libraries prepared from cell-free DNA (cfDNA)?
    16. Can EM-seq libraries be prepared from FFPE DNA?
    17. What levels of conversion are typical with the control DNAs supplied in the EM-seq kit?
    18. Can enzymatically fragmented DNA be used as input material for EM-seq™?
    19. Are Sample Sheets available for use with the NEBNext® Multiplex Oligos for EM-seq?
    20. Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction?
    21. Are dual indexed libraries compatible with single end sequencing?
    22. Is it normal if the FE(II) solution from the EM-seq product is yellow or a color change is observed?
    23. What percentage of PhiX is needed for EM-seq libraries?