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商品详细NEB/NEBNext FFPE DNA Repair v2 Module/24 reactions/E7360S
NEB/NEBNext FFPE DNA Repair v2 Module/24 reactions/E7360S
NEB/NEBNext FFPE DNA Repair v2 Module/24 reactions/E7360S
商品编号: E7360S
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产地: 美国(厂家直采)
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联系Q Q: 3392242852
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商品介绍

The NEBNext FFPE DNA Repair v2 Module contains enzymes and buffers that have been optimized to repair FFPE DNA in next generation sequencing workflows in a streamlined manner.The NEBNext FFPE DNA Repair v2 Module improves upon the performance of the original NEBNext FFPE DNA Repair Mix:

  • Higher repair efficiency with FFPE DNA
  • A more streamlined workflow for NGS library preparation
  • A more convenient reaction buffer containing all required buffer components for both efficient FFPE DNA repair and downstream end repair and dA-tailing.
  • No cleanup is required between repair and library prep, through the use of Thermolabile Proteinase K.

Table 1: Types of FFPE DNA damage and their ability to be repaired by the NEBNext FFPE DNA Repair v2 Module

FFPE DAMAGE TYPE REPAIRED BY THE FFPE DNA REPAIR v2 MODULE
Deamination of cytosine to uracil Yes
Nicks and gaps Yes
Oxidized bases Yes
Blocked 3´ ends Yes
DNA fragmentation No
DNA-protein crosslinks No
Figure 1: Workflow demonstrating the use of the NEBNextFFPE DNA Repair v2 Module with the NEBNext Ultra™ II DNA Library Prep Kit for Illumina®
Figure 2: The NEBNext FFPE DNA Repair v2 Module enables robust library preparation from a broad range of FFPE DNA sample qualities.
Libraries were prepared with 25 ng of Covaris®acoustic-sheared FFPE DNA samples of different qualities and tissue sources. The NEBNext FFPE DNA Repair v2 Module was used, followed by NEBNext Ultra II DNA library preparation (NEB #E7645) with 9 PCR cycles. Libraries were quantified using the Agilent® HS D1000 TapeStation®. The NEBNext FFPE DNA Repair v2 Module improves the yield of FFPE libraries by varying degrees depending on the quality and damage types present in the input DNA. Error bars indicate the standard deviation of two replicates for each library sample.
Figure 3: The NEBNext FFPE DNA Repair v2 Module enables robust library preparation from 5 to 250 ng of FFPE DNA input using both NEBNext hairpin and Unique Dual Index UMI Adaptors
Libraries were prepared with 5, 50, or 250 ng of three different quality normal liver FFPE DNA samples comparing treatment with and without the NEBNext FFPE DNA Repair v2 Module. The NEBNext Ultra II DNA Library Preparation Kit (NEB #E7645) was used with (a) the NEBNext hairpin adaptor and 11, 8 and 6 PCR amplification cycles for 5, 50 and 250 ng inputs, respectively, and (b) the NEBNext Unique Dual Index UMI DNA Adaptors (NEB #E7395) and 11, 8 and 6 PCR cycles for 5, 50 and 250 ng inputs, respectively. All libraries were quantified using the Agilent HS D1000 Tapestation and the average yield of 2 libraries (5 and 50 ng) and 1 replicate (250 ng) was plotted. Error bars indicate standard deviation for the 5 and 50 ng library replicates. The NEBNext FFPE DNA Repair v2 Module is compatible with 5 to 250 ng inputs of FFPE DNA and different NEBNext adaptors.
Figure 4: The NEBNext FFPE DNA Repair v2 Module improves library quality metrics including mapping rate, properly paired reads, and chimeric reads
Libraries were prepared with 50 ng of three different quality normal liver FFPE DNA samples in triplicate, either untreated or treated with the NEBNext FFPE DNA Repair v2 Module before library preparation using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Libraries were sequenced on the Illumina NextSeq®500. Paired-end reads were downsampled to 1 million reads and mapped to the GRCh38 human reference (RefSeq 884148) using Bowtie2 (v2.3.2). Mapped reads were analyzed with MarkDuplicates (v1.56.0) and Picard SAM/BAM alignment summary metrics (v1.56.0). Treatment with the NEBNext FFPE DNA Repair v2 Module increases the mapping rate and decreases the level of non-properly paired and chimeric reads.
Figure 5: The NEBNext FFPE DNA Repair v2 Module repairs abundant cytosine deamination artifacts present in FFPE DNA samples
50 ng samples of two different normal liver FFPE DNAs (DIN 2.0 and DIN 1.8) were either untreated or treated in duplicate with the NEBNext FFPE DNA Repair v2 Module, followed by library preparation using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Libraries were sequenced on the Illumina NextSeq®500. Paired-end reads were downsampled to 1 million reads and mapped to the GRCh38 human reference (RefSeq 884148) using Bowtie2 (v2.3.2). Mapped reads were analyzed with MarkDuplicates (v1.56.0) and dCdT (CT) mutations were analyzed using Tasmanian (V0.1.3) according to the equation MAX(([C T] - [G A]) / ([NC]+[NG]), EXP(-10)). (a) The frequency of artifactual CT mutations was plotted as a function of read position (0 to 75 bp) in read 1 and read 2. The abundance and positional bias of these artifacts were reduced in two different FFPE DNA samples when treated with the NEBNext FFPE DNA Repair v2 Module. (b) Artifactual mutations identified in read 2 were quantified as an overall frequency. Two replicates for each condition are shown.
Figure 6: The NEBNext FFPE DNA Repair v2 Module repairs oxidative damage in FFPE and non-FFPE DNA samples
A. Libraries were prepared in duplicate with 25 ng of two different normal FFPE DNA samples of different qualities and tissue sources, comparing untreated to treated with the NEBNext FFPE DNA Repair v2 Module. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645), sequenced on the Illumina NextSeq® 500, downsampled to 2 million reads, mapped to the GRCh38 human reference genome using Bowtie2 (v2.3.2). Mapped reads were analyzed by MarkDuplicates (v1.56.0) and Tasmanian (V0.1.3). The frequency of artifactual dGdT (GT) mutations in read 1 are shown for each library replicate. dGdT mutations were calculated according to the equation MAX(([G T] - [C A]) / ([NG]+[NC]), EXP(-10)).B. Libraries were prepared in duplicate using 100 ng of human genomic DNA Covaris-sheared in either water* or Tris-EDTA pH 8.0, the NEBNext FFPE DNA Repair v2 Module, and the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Libraries were sequenced on the Illumina NextSeq 500, downsampled to 2 million reads and analyzed for GT mutation frequency as above. *Note: Shearing DNA in water is not recommended by the manufacturer (Covaris) but was used to generate a substrate containing oxidative damage.
Figure 7: The NEBNext FFPE DNA Repair v2 Module improves library yield compared to treatment with UDG enzyme
Libraries were prepared in duplicate using 50 ng FFPE DNA input from 4 different normal FFPE DNAs of different tissue sources and qualities (FFPE #1-3 with DIN 2.0 and FFPE #4 with DIN 6.7). Libraries were either untreated, treated with the NEBNext FFPE DNA Repair v2 Module, or treated with UDG enzyme (NEB #M0280) before preparing libraries with the NEBNext Ultra II DNA Library Preparation Kit for Illumina (NEB #E7645). While UDG enzyme can eliminate dU bases from input DNA, the NEBNext FFPE DNA Repair v2 Module can fully repair these damage sites and improve the final library yield (Figures 2, 3, 7) and metrics (Figure 4).
Figure 8: The NEBNext FFPE DNA Repair v2 Module reduces false positives in somatic variant calling arising from cytosine deamination artifacts
Libraries were prepared from 100 ng of four different FFPE DNA samples of varying qualities and tissue sources in duplicate using the NEBNext FFPE DNA Repair v2 Module or NEBNext FFPE DNA Repair Mix (NEB #M6630), the NEBNext Unique Dual Index UMI Adaptors (NEB #E7395), the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) and 10 PCR cycles. Libraries were captured using a custom cancer hotspot panel (Twist Bioscience®) and sequenced on the Illumina NextSeq 2000. All fastq files were downsampled to the same number of reads (2 X 18 million reads fresh frozen and 2 X 13 million for FFPE). Paired reads were trimmed with fastp (version 0.20.0) and mapped with BWA mem (0.7.17). Markduplicate was processed in picard (2.20.6) with UMI information. UMI information was processed in fgbio (0.8.1) to obtain consensus sequence reads. The final bam files with UMI-based consensus reads were used for somatic variant calling with strelka2 (2.9.10). Somatic variant calls are plotted according to substitution type for each condition. NEBNext FFPE DNA Repair v2 improves the efficiency of cytosine deamination repair (CT/GA) and effectively repairs oxidative damage (GT/CA).
This product is related to the following categories:
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。
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