Next Direct® Genotyping Solution |NEB酶试剂 New England Biolabs

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The NEBNext Direct Genotyping Solution combines highly multiplexed, capture-based enrichment with maximum efficiency next-generation sequencing to deliver cost-effective, high-throughput, genotyping for a wide variety of applications. Applicable for marker ranges spanning 100-5,000 markers, pre-capture multiplexing of up to 96 samples combined with dual indexed sequencing allows over 3.8 million genotypes in a single Illumina® sequencing run.

 

Figure 1: NEBNext Direct Genotyping Solution workflow 

Next Direct® Genotyping Solution |

Figure 2: Passing filter reads across 96 pooled samples

Next Direct® Genotyping Solution |

Passing filter reads across 96 tomato DNA samples that were enriched using a genotyping panel consisting of 2,309 publicly available SolCAP markers and the NEBNext Direct Genotyping Solution. 25 ng of purified tomato DNA was used for each sample. Samples were index-tagged and pooled prior to hybridization and libraries were sequenced on an Illumina® MiSeq® with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.
Figure 3: NEBNext Direct Genotyping Solution demonstrates similar coverage across 96 pooled samples

Next Direct® Genotyping Solution |

Mean SNP coverage of 2,309 SolCAP markers across 96 samples. 25 ng of purified tomato DNA was used for each sample. Samples were index-tagged and pooled prior to hybridization and Libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.
Figure 4: Specificity of enrichment of NEBNext Direct Genotyping Solution across 96 pooled samples

Next Direct® Genotyping Solution |

The percent of passing filter reads mapping to targeted regions demonstrates high specificity across 96 multiplexed samples using the NEBNext Direct Genotyping Solution. 25 ng of purified tomato DNA was used as input for each sample. Samples were index-tagged and pooled prior to hybridization and Libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.
Figure 5: Mean Coverage across 2309 markers within a single sample
Next Direct® Genotyping Solution |
Histogram of coverage across each of the 2,309 SolCAP markers demonstrates evenness of enrichment across targets and coverage levels sufficient for genotyping calls. These data represent enrichment of a single tomato sample pooled with 95 others prior to hybridization. 25 ng of purified tomato DNA was used for each sample. Samples were index-tagged and pooled prior to hybridization and libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.

Reagents Supplied for E9500

The following reagents are supplied with this product:

  Store at (°C) Concentration
NEBNext Direct® GS Fragmentation Buffer -20  
NEBNext Direct® GS Fragmentation Enzyme Mix -20  
NEBNext Direct® GS Indexed 5′ Adaptor Plate -20  

NEBNext Direct® GS 5′ Adaptor Ligation Master Mix

 -20  
NEBNext Direct® GS Stop Solution 4  
NEBNext Direct® GS Sample Purification Beads RT  

Reagents Supplied for E9530

The following reagents are supplied with this product:

  Store at (°C) Concentration
NEBNext Direct® GS Baits -20 (pouch)  
NEBNext Direct® GS Hybridization Additive -20  
NEBNext Direct® GS 3′ Blunting Enzyme -20  
NEBNext Direct® GS 3′ Adaptor -20  
NEBNext Direct® GS Ligase -20  
NEBNext Direct® GS Specificity Enhancer Enzyme Mix -20  
NEBNext Direct® GS Q5 Master Mix -20  
NEBNext Direct® GS Index Primer Mix DGS01 -20  
NEBNext Direct® GS Index Primer Mix DGS02 -20  
NEBNext Direct® GS Index Primer Mix DGS03 -20  
NEBNext Direct® GS Index Primer Mix DGS04 -20  
NEBNext Direct® GS Index Primer Mix DGS05 -20  
NEBNext Direct® GS Index Primer Mix DGS06 -20  
NEBNext Direct® GS Index Primer Mix DGS07 -20  
NEBNext Direct® GS Index Primer Mix DGS08 -20  
NEBNext Direct® GS Pre-Hyb Sample Purification Beads RT  
NEBNext Direct® GS Bead Wash 1 (BW1) RT  
NEBNext Direct® GS Post-PCR Sample Purification Beads RT  
NEBNext Direct® GS TE Buffer 4  
NEBNext Direct® GS Hybridization Buffer 4  
NEBNext Direct® GS Streptavidin Beads 4  
NEBNext Direct® GS Bead Prep Buffer 4  
NEBNext Direct® GS Hybridization Wash (HW) 4  
NEBNext Direct® GS Bead Wash 2 (BW2) 4  
NEBNext Direct® GS 3′ Blunting Buffer 4  
NEBNext Direct® GS 3′ Adaptor Ligation Buffer 4  

 

产品类别:
Automation for NEBNext® NGS Library Prep Products

  • 优势和特性

    应用特性

    • The most efficient targeted genotyping-by-sequencing solution available
      • Pre-capture multiplexing of up to 96 samples per enrichment
      • Flexibility from 100’s to 1,000’s of markers per sample
      • Dual indexing plus Unique Molecular Identifier (UMI)
      • Optimized bait design captures both DNA strands
    • Unique and rapid, capture-based enrichment for optimal sequencing performance
      • Highly specific enrichment, > 90% of reads mapping to targets
      • Coverage uniformity – sequence less and avoid over- and under-representation
      • Similar performance from DNA across CTAB and column-based extraction procedures
    • Fast and streamlined, automation-friendly workflow, with minimal handling steps and hands-on time’
      • Pooled capture eliminates redundancy
      • Single-day workflow
      • Automation friendly

操作说明、说明书 & 用法

  • 操作说明

    1. Protocol for use with NEBNext Direct Genotyping Solution (NEB #E9500 and #E9530)
    2. Guidelines for Using the Single-use, Indexed 5′ Adaptor Plate (NEB #E9500 and #E9530)
    3. Index Adaptor and Index Primer Pooling Guidelines (NEB #E9500 and #E9530)
    4. Guidelines for Running Samples on the Illumina MiSeq (NEB #E9500 and #E9530)
    5. Guidelines for Bioinformatic Processing of Sequencing Data (NEB #E9500 and #E9530)

  • 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。

    • manualE9500_E9530

  • 使用指南

    • bcl2fastq – Guildelines for E9500 and E9530
    • bcl2fastq – Sample Sheet for E9530-768
    • NEBNext Direct GS Barcodes
    • Picard – Guidelines for Sample Metadata for E9500 and E9530
    • Picard – Sample Metadata for E9530
    • Sample Sheet – E9530 – E9530
    • Sample Sheet – Guidelines for E9500B and E9530B

  • 应用实例

    • Fully Automated NEBNext® Direct Genotyping Solution using Beckman Coulter® Life Sciences Biomek i7 Workstation

FAQs & 问题解决指南

  • FAQs

    1. How many times can the -20°C reagents supplied in the NEBNext Direct® Genotyping Solution be frozen and thawed? 
    2. How many times can the 4°C reagents be brought to room temperature?
    3. My NEBNext Direct Genolytyping Solution 4°C box accidentally froze, can I still use it?
    4. I accidentally stored the Sample Purification Beads and Bead Wash 1 (BW1) at 4°C. Can I still use them?
    5. What is the shelf life of the NEBNext Direct Genotyping Solution?
    6. What method do you recommend for extracting genomic DNA and FFPE DNA before use with the NEBNext Direct Genotyping Solution?
    7. What is the recommended method for quantitating my input DNA?
    8. If my input amount is low, should I dilute the adaptors?
    9. What type and how much starting material do I need to use when preparing libraries using the NEBNext Direct Genotyping Solution?
    10. Which magnets (2 ml and 200 µl sizes) do you recommend?
    11. I used Bead Wash 1 (BW1) when I should have used Bead Wash 2 (BW2) during the Post-reaction Wash. Can I do another wash with Bead Wash 2 (BW2) to save my sample?
    12. I used Bead Wash 2 (BW2) when I should have used Bead Wash 1 (BW1) during the Post-reaction Wash. What should I do?
    13. Does incomplete removal of Bead Wash 1 prior to adding Bead Wash 2 inhibit the next step?
    14. Can I use any Q5® High-Fidelity DNA Polymerase formulation with this kit?
    15. If I have adaptor dimer in my finished library, can I perform another bead cleanup to remove it?

    16. How many/ few samples can I pool together for my sequencing run?

    17. Are blocking oligos required for this kit?
    18. Is the NEBNext Direct Genotyping Solution available in bulk or customized format?