标签归档：ultrashear

Next UltraShear® |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

View or download extensive performance data in our Data Supplement.

Enzymatic methods for DNA fragmentation in NGS workflows enable streamlined protocols, improved performance and scalability. However, specialized fragmentation reagents are required for samples for methylation analysis, to ensure that methylation marks are not removed, and for FFPE DNA. NEBNext UltraShear is a novel enzyme mix designed for fragmentation of these sample types that has a fast workflow and improves library preparation and sequencing metrics for DNA methylation studies and FFPE DNA.
NEBNext UltraShear is compatible with NEBNext Enzymatic Methyl-seq (EM-seq) (NEB #E7120).

NEBNext UltraShear in FFPE DNA sequencing workflows:

  • Improved usable reads
  • Lower artificial mutation frequency

NEBNext UltraShear for methylated DNA sequencing e.g., EM-seq

  • Higher library yields
  • Improved sequencing metrics
  • Improved CpG Coverage

Note that for high quality genomic DNA library prep with enzymatic fragmentation, we recommend NEBNext Ultra II FS DNA Library Prep for Illumina (NEB #E7805, #E6177).

 

Figure 1: NEBNext UltraShear increases EM-seq™ library yields

Next UltraShear® |

200 ng, 50 ng and 10 ng of NA12878 DNA spiked with control DNA (CpG methylated pUC19 DNA and unmethylated lambda DNA) were fragmented by either NEBNext UltraShear (20 minutes at 37°C) or Covaris® ME220 (350 bp protocol) followed by EM-seq library preparation. Library yields were quantified using Agilent® TapeStation® with the High Sensitivity D1000 ScreenTape®. EM-seq libraries fragmented by NEBNext UltraShear have higher yields than Covaris for the same number of PCR cycles for each input (200 ng = 4 cycles; 50 ng = 6 cycles; 10 ng = 8 cycles).

Figure 2: Improved CpG coverage in EM-seq libraries produced using NEBNext UltraShear

Next UltraShear® |

200 ng, 50 ng and 10 ng of NA12878 DNA spiked with control DNA (CpG methylated pUC19 DNA and unmethylated lambda DNA) was fragmented by either NEBNext UltraShear (20 minutes at 37°C) or Covaris ME220 (350 bp protocol) followed by EM-seq library preparation. Technical replicates were generated for each input amount. All libraries were sequenced on the same flowcell of an Illumina® NovaSeq® 6000 (2 x 100 bases). 725 million reads were sampled (seqtk) from each library for methylation analysis. Reads were adapter trimmed (fastp), aligned to the GRCh38 reference (bwa-meth), and duplicate marked (Picard MarkDuplicates) before calling methylation using MethylDackel. NEBNext UltraShear and Covaris fragmentation used ahead of the EM-seq workflow yielded a similar number of CpGs (~54 million) at minimum 1X coverage. At minimum 10X coverage, more CpGs are identified when NEBNext UltraShear is used, due to improved library diversity and coverage evenness.

Figure 3: NEBNext UltraShear with FFPE DNA improves usable reads

Next UltraShear® |

FFPE DNA was fragmented using NEBNext UltraShear (15 minutes at 37°C), Covaris ME220, Kapa EvoPlus® Kit, Kapa HyperPlus® Kit, Agilent SureSelect® Enzymatic Fragmentation Kit, NEBNext dsDNA Fragmentase® or NEBNext Ultra II FS. All samples were fragmented according to the respective protocols. Fragmentation with NEBNext dsDNA Fragmentase, Kapa kits and the Agilent kit was followed by a bead cleanup and library construction using the NEBNext Ultra II DNA library Prep Kit for Illumina. NEBNext UltraShear and Covaris-sheared samples were followed directly by use of the NEBNext Ultra II DNA Library Prep Kit for Illumina, and NEBNext Ultra II FS samples followed the recommended protocol for library prep. Each library was sequenced using the Illumina NextSeq® 500. 2 million (2 x 76 base) reads were used for this analysis. Reads were aligned to GRCh38 with Bowtie2 and analyzed using samtools flagstats and Picard CollectAlighmentSummaryMetrics. Percent of usable reads (mappable, proper pairs, and non-duplicates reads) were measured for each library and usable reads were averaged for technical replicates (bars represent error between two technical replicates) for all fragmentation methods. FFPE DNA libraries fragmented with NEBNext UltraShear had the highest percent of usable reads and had similar percent usable reads as high-quality DNA libraries (high-quality DNA had a comparable percent of usable reads across all fragmentation methods ≥ 96%; data not shown).

Figure 4: NEBNext UltraShear with FFPE DNA reduces artificial mutations

Next UltraShear® |

FFPE DNA was fragmented using NEBNext UltraShear (15 minutes at 37°C), Covaris ME220, Kapa EvoPlus Kit, Kapa HyperPlus Kit, Agilent SureSelect Enzymatic Fragmentation Kit, NEBNext dsDNA Fragmentase or NEBNext Ultra II FS. All samples were fragmented according to the recommended protocols. Fragmentation with NEBNext dsDNA Fragmentase, Kapa® kits and the Agilent kit was followed by a bead cleanup and NEBNext Ultra II DNA library Prep Kit for Illumina. NEBNext UltraShear and Covaris-sheared samples were followed directly by use of the NEBNext Ultra II DNA Library Prep Kit for Illumina, and NEBNext Ultra II FS samples followed the recommended protocol for library prep. Each library was sequenced using the Illumina NextSeq 500. 2 million (2 x 76 base) reads were used for this analysis. Reads were aligned to GRCh38 with Bowtie2. Artificial C to T mutations were calculated with Tasmanian tool for read 1 and 2 and averaged for technical replicates (bars represent error between two technical replicates). The libraries fragmented with NEBNext UltraShear resulted in the lowest C to T artificial mutation frequency compared to other fragmentation methods for FFPE DNA both reads (R1= Read 1 and R2= Read 2).

Figure 5: NEBNext UltraShear fragments high-quality genomic DNA in a time-dependent manner

Next UltraShear® |

50 ng human DNA (NA12878) was fragmented for 5–45 minutes at 37°C followed by 15 minutes at 65°C. Fragmentation occurs during the 37°C incubation step of NEBNext UltraShear. The average fragmentation size and pattern (High Sensitivity D5000 ScreenTape on Agilent TapeStation) is based on fragmentation time.

产品类别:
FFPE DNA Products,
DNA Fragmentation & RNA Fragmentation 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
    • M7634S     -20    
        NEBNext UltraShear® M7634SVIAL -20 1 x 0.096 ml Not Applicable
        NEBNext UltraShear™ Reaction Buffer B9042SVIAL -20 1 x 0.336 ml Not Applicable
        500 mM DTT B1079SVIAL -20 1 x 0.048 ml Not Applicable
    • M7634L     -20    
        NEBNext UltraShear® M7634LVIAL -20 1 x 0.384 ml Not Applicable
        NEBNext UltraShear™ Reaction Buffer B9042LVIAL -20 2 x 0.672 ml Not Applicable
        500 mM DTT B1079LVIAL -20 1 x 0.192 ml Not Applicable

  • 特性和用法

    需要但不提供的材料

    • 1X TE (10 mM Tris pH 8.0, 1 mM EDTA)
    • 0.2 ml thin wall PCR tubes
    • Magnetic rack/stand (NEB #S1515S; Alpaqua® #A001322 or equivalent)
    • PCR machine
    • Vortex
    • Microcentrifuge
    • Bioanalyzer®, TapeStation® or other fragment analyzer and associated consumables
    • 80% Ethanol

    For use with NEBNext UltraShear Protocol:

    • SPRIselect™ Reagent Kit (Beckman Coulter, Inc. #B23317), AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or Monarch®® PCR & DNA Cleanup Kit (NEB# T1030S/L) are recommended for Section 1.

    For use with NEBNext Ultra II End Repair/dA-Tailing Module Protocol:

    • NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546S/L) for Section 2.
    • Recommended Material Not Included: NEBNext Ultra II Ligation Module (NEB #E7595) and NEBNext Multiplex Oligos (www.neb.com/oligos).

    For use with NEBNext Enzymatic Methyl-seq Protocol:

    • NEBNext Enzymatic Methyl-seq Kit (NEB #E7120S/L) for Section 3.
    • Formamide (Sigma #F9037-100 ml) or optional 0.1 N NaOH. Formamide is preferred. If using NaOH, please see NEBNext Enzymatic Methyl-seq Kit (NEB #E7120) FAQs 
    • Nuclease-free

  • 相关产品

    相关产品

    • NEBNext® 酶学转化法甲基化建库试剂盒
    • NEBNext® 甲基化建库酶学转化法模块
    • E7360 NEBNext FFPE DNA 修复模块 v2

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find guidelines and protocols for using NEBNext UltraShear, including in conjunction with NEBNext Enzymatic Methyl-seq (EM-seq™)?

  • 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。

    • manualM7634

工具 & 资源

  • Web 工具

    • NEBNext Selector

FAQs & 问题解决指南

  • FAQs

    1. Is NEBNext UltraShear™ the same as NEBNext® Ultra™ II FS or NEBNext dsDNA Fragmentase®?
    2. Do you really need to vortex NEBNext UltraShear™?
    3. For EM-seq™ workflows, what are the recommendations for fragmenting already-fragmented DNA, low integrity DNA and/or FFPE DNA with NEBNext UltraShear™?
    4. Following the fragmentation step of the NEBNext UltraShear™ protocol, can the reactions be stored at -20˚C?
    5. Do you recommend NEBNext UltraShear™ for high-sensitivity, low error rate DNA library preparation?

Next Ultrashear® FFPE DNA Library Prep Kit |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

View or download extensive performance data in our Data Supplement.

FFPE DNA is often compromised in quality and quantity and is challenging for library prep and sequencing, including in target enrichment workflows that require high amounts of library input. The NEBNext UltraShear FFPE DNA Library Prep Kit addresses these challenges and improves ease of workflow and scalability:

  • Repairs FFPE-induced damage using the NEBNext FFPE DNA Repair Mix v2
  • Incorporates specialized enzymatic DNA fragmentation using NEBNext UltraShear
  • Uses NEBNext Ultra II library prep reagents in a protocol optimized for FFPE DNA
  • Robustly amplifies libraries using NEBNext MSTC™ FFPE Master Mix
  • High library yields, sufficient for input into target enrichment workflows
  • User-friendly workflow with minimal hands-on time and multiple safe stopping points

The NEBNext UltraShear FFPE DNA Library Prep Kit reduces damage from deamination and oxidation due to the fixation, storage, and extraction process for FFPE DNA samples, thereby reducing false positive variant calls.

The combination of specialized enzymatic FFPE DNA fragmentation and repair with optimized reagents and protocols provides superior performance with this challenging sample type.

For FFPE DNA library prep without enzymatic fragmentation, the NEBNext FFPE DNA Library Prep Kit (NEB #E6650) is available.

FIGURE 1: NEBNext UltraShear FFPE DNA Library Prep Kit workflow overview

Next Ultrashear® FFPE DNA Library Prep Kit |

The NEBNext UltraShear FFPE DNA Library Prep Kit has a streamlined workflow with minimal hands-on time across a range of inputs from 5-250 ng. The protocol has been optimized for the user to safely store the reaction after any step in the workflow overnight at -20°C without affecting library yield or quality.
FIGURE 2: The NEBNext UltraShear FFPE DNA Library Prep Kit enables robust library preparation from a range of sample inputs and quality

Next Ultrashear® FFPE DNA Library Prep Kit |

Libraries were prepared from 5, 50, or 250 ng of normal tissue FFPE DNA ranging in quality from DNA Integrity Number (DIN) 1.8 to 6.8, with the indicated PCR cycles. Libraries were prepared in triplicate for 5 ng and 50 ng input and 1 replicate for 250 ng. Each bar represents the average of triplicates with error bars indicating standard deviation for the 5 and 50 ng inputs. Robust library yields were obtained across sample qualities and input amounts. Most target enrichment workflows require a 200 ng library for hybrid capture input. Sufficient library yield can be obtained using a minimum of 50 ng FFPE DNA with the NEBNext UltraShear FFPE DNA Library Prep Kit.    

FIGURE 3: The NEBNext UltraShear FFPE DNA Library Prep Kit improves library quality

Next Ultrashear® FFPE DNA Library Prep Kit |    

Libraries were prepared in duplicate from 100 ng of low quality, normal tissue FFPE DNA (DIN 1.8) and 9 PCR cycles, using the NEBNext UltraShear™ FFPE DNA Library Prep Kit. Results were compared to other enzymatic fragmentation-based library prep kits that have been validated for use with FFPE samples, using each vendor’s own recommended adaptors (IDT® xGen® EZ UNI, Kapa EvoPlus® Library Prep Kit, QuantaBio® sparQ DNA Library Prep Kit, and Twist Library Preparation EF 2.0 kit). Libraries were sequenced on the Illumina® NovaSeq®6000 (2 x 100 base reads) and downsampled to 5 million paired-end reads. Reads were mapped using Bowtie2 (version 2.3.2.2) to the GRCh38 reference and duplicates were marked using Picard MarkDuplicates (version 1.56.0). Library quality metrics were assessed using Picard Alignment Summary Metrics (version 1.56.0). The level of foldback reads was calculated using Seq_frag_remap (version 0.2). The NEBNext UltraShear FFPE DNA Library Prep Kit improves library quality by reducing the percentage of unmapped, chimeric, non-properly paired, and foldback reads.
FIGURE 4: The NEBNext UltraShear FFPE DNA Library Prep Kit reduces sequencing artifacts

Next Ultrashear® FFPE DNA Library Prep Kit |

Libraries were prepared in duplicate from 100 ng of low quality, normal tissue FFPE DNA (DIN 1.8) and 9 PCR cycles, using the NEBNext UltraShear FFPE DNA Library Prep Kit. Results were compared to other enzymatic fragmentation-based library prep kits that have been validated for use with FFPE samples, with each vendor’s own recommended adaptors (IDT xGen EZ UNI, Kapa EvoPlus Library Prep Kit, QuantaBio sparQ DNA Library Prep Kit, and Twist Library Preparation EF 2.0 kit). Libraries were sequenced on the Illumina NovaSeq 6000 (2 x 100 base reads) and downsampled to 5 million paired-end reads. Reads were mapped using Bowtie2 (version 2.3.2.2) to the GRCh38 reference and duplicates marked using Picard MarkDuplicates (version 1.56.0). The average frequency of C→T mutations at each C position (A) and G→T mutations at each G position (B) in Read 1 and 2 was calculated for two technical replicates using Tasmanian (version 1.0.7). C→T mutations arising from cytosine deamination and G→T mutations arising from oxidative damage in low quality FFPE DNA are effectively repaired by the NEBNext FFPE DNA Repair v2 Mix in the NEBNext UltraShear FFPE DNA Library Prep Kit. Other kits show a high level of C→T and G→T artifacts in low quality FFPE DNA due to a lack of DNA damage repair.

FIGURE 5: The NEBNext UltraShear FFPE DNA Library Prep Kit enables superior on-target coverage in hybrid capture sequencing

Next Ultrashear® FFPE DNA Library Prep Kit |

Libraries were prepared in duplicate from 100 ng of low quality, normal tissue FFPE DNA (DIN 1.8) and 9 PCR cycles, using the NEBNext UltraShear™ FFPE DNA Library Prep Kit. Results were compared to other enzymatic fragmentation-based library prep kits that have been validated for use with FFPE samples, with each vendor’s own recommended adaptors (IDT xGen EZ UNI, Kapa EvoPlus Library Prep Kit, QuantaBio sparQ DNA Library Prep Kit, and Twist Library Preparation EF 2.0 kit). The full library yield was used in singleplex target enrichment with a custom cancer panel (Twist Bioscience®) and libraries were sequenced on the Illumina NovaSeq 6000 (2 x 100 base reads). 15 million paired-end reads were trimmed with Fastp (version 0.20.0) and mapped with BWA mem (version 0.7.17) to the T2T reference. Duplicates were marked using Picard MarkDuplicates (version 2.20.6) with UMI. Target enrichment quality metrics were assessed using Picard HS Metrics (version 2.18.29). The improved yield, coverage, and fraction of usable reads observed in NEBNext UltraShear FFPE DNA Library Prep Kit whole genome sequencing (WGS) libraries correlates to improved coverage, on-target rate, and coverage uniformity in target enrichment libraries.

产品类别:
FFPE DNA 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
    • E6655S     Multi-temperature    
        NEBNext Ultrashear® FFPE DNA Library Prep Kit E6655-2 -20    
        NEBNext® FFPE DNA Repair Mix v2 E7361AVIAL -20 1 x 0.048 ml Not Applicable
        NEBNext® Thermolabile Proteinase K E7362AVIAL -20 1 x 0.048 ml Not Applicable
        NEBNext UltraShear™ Reaction Buffer E6657AVIAL -20 1 x 0.168 ml Not Applicable
        NEBNext UltraShear™ E6656AVIAL -20 1 x 0.096 ml Not Applicable
        500mM DTT E6658AVIAL -20 1 x 0.048 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
        NEBNext® MSTC FFPE Master Mix E6651AVIAL -20 1 x 0.6 ml 2 X
        NEBNext® Sample Purification Beads E6652S 25    
        NEBNext® Sample Purification Beads E6652AVIAL 25 1 x 4.3 ml Not Applicable
    • E6655L     Multi-temperature    
        NEBNext Ultrashear® FFPE DNA Library Prep Kit E6655-3 -20    
        NEBNext® FFPE DNA Repair Mix v2 E7361AAVIAL -20 1 x 0.192 ml Not Applicable
        NEBNext® Thermolabile Proteinase K E7362AAVIAL -20 1 x 0.192 ml Not Applicable
        NEBNext UltraShear™ Reaction Buffer E6657AAVIAL -20 1 x 0.672 ml Not Applicable
        NEBNext UltraShear™ E6656AAVIAL -20 1 x 0.384 ml Not Applicable
        500mM DTT E6658AAVIAL -20 1 x 0.192 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
        NEBNext® MSTC FFPE Master Mix E6651AAVIAL -20 2 x 1.2 ml 2 X
        NEBNext® Sample Purification Beads E6652L 25    
        NEBNext® Sample Purification Beads E6652AAVIAL 25 1 x 17 ml Not Applicable

  • 特性和用法

    需要但不提供的材料

    • 80% Ethanol
    • Nuclease-free Water
    • 0.1X TE (1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA)
    • DNase-, RNase-free PCR strip tubes
    • NEBNext Multiplex Oligos for Illumina® (www.neb.com/oligos)
    • Magnetic rack/stand (NEB #S1515S, Alpaqua® cat. #A001322, or equivalent)
    • Thermal cycler
    • Agilent® Bioanalyzer® or TapeStation® and associated reagents and consumables
    • Adaptor Dilution Buffer NEB #B1430S or NEBNext Unique Dual Index UMI Adaptor Dilution Buffer supplied with NEBNext Unique Dual Index UMI Adaptor DNA Sets (NEB #E7395/E7874/E7876/E7878)

  • 相关产品

    相关产品

    • e6650nebnext-ffpe-dna-library-prep-kit
    • NEBNext® 多样本接头引物试剂盒 1（96 种 Unique 双端 Index 引物）
    • NEBNext® 多样本接头引物试剂盒 2（96 种 Unique 双端 Index 引物）
    • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3)

操作说明、说明书 & 用法

  • 操作说明

    1. Where can I find guidelines and protocols for use of the NEBNext UltaShear FFPE DNA Library Prep Kit (NEB #E6655)?

  • 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。

    • manualE6655

工具 & 资源

  • 选择指南

    • NEBNext® Multiplex Oligos Selection Chart

FAQs & 问题解决指南

  • FAQs

    1. What sample types can I use with the NEBNext UltraShear FFPE DNA Library Prep Kit?
    2. How much starting material is required for the NEBNext UltraShear FFPE DNA Library Prep Kit?
    3. When preparing samples using the NEBNext UltraShear FFPE DNA Library Prep Kit, can my input DNA be in EDTA-containing solutions?
    4. Do I really need to vortex the NEBNext UltraShear enzyme mix before use?
    5. What fragmentation time should be used with the NEBNext UltraShear FFPE DNA Library Prep Kit for FFPE DNA samples?
    6. Is size selection recommended?
    7. What are the safe stopping points in this protocol?
    8. Which NEBNext Multiplex Oligos can be used with the NEBNext UltraShear FFPE DNA Library Prep Kit?
    9. What input amount (nanograms) is recommended for standard versus target enrichment library prep with FFPE DNA samples?
    10. Can I use this NEBNext kit with adaptors and primers from vendors other than NEB?
    11. Will treating my DNA with the FFPE DNA Repair Mix v2 as part of the NEBNext UltraShear FFPE DNA Library Prep Kit hurt my downstream reaction?
    12. Does the FFPE DNA Repair v2 Mix repair DNA-protein crosslinks?
    13. Does the FFPE DNA Repair v2 Mix fix blocked 3′ ends?
    14. Can the FFPE DNA Repair v2 Mix repair damage in both single- and double-stranded DNA? Or, does it require double stranded DNA as a template?
    15. If I had a DNA template with mutation sites (i.e. 8-oxoguanine or deaminated cytosines) that are directly adjacent to each other on opposite strands would treatment with the FFPE DNA Repair v2 Mix cause a double strand nick/break?
    16. What gap lengths can be repaired with the FFPE DNA Repair v2 Mix?