Next Gen Sequencing

Protocols and Support

Additional Resources

xGen® Lockdown® Probes

Target Capture Probe Pools for NGS

     
Use xGen Lockdown probe pools to generate customized target capture panels for enrichment of your targets of interest or to enhance the performance of existing panels. Both predesigned (xGen Predesigned Gene Capture Pools and Plates) and custom probes (xGen Custom Target Capture Probes) are available.

Benefits

  • Achieve higher performance with individually synthesized and quality-controlled capture probes
  • Develop customized panels with xGen Predesigned Gene Capture Pools or Custom xGen Custom Target Capture Probes
  • Start your project faster with a 7–10 day turnaround time

Features

  • All probes are synthesized as Ultramer® Oligonucleotides, using our highest coupling efficiency synthesis, and contain a 5' biotin modification
  • Probes are individually assessed by mass spectrometry for QC
  • xGen Predesigned Gene Capture Pools are available for any human RefSeq coding gene, provided premixed or in individual wells for selective mixing
  • Up to 2000 probes per pool; larger probe pools by request
  • Probes are shipped in 7–10 days

See the Overview Tab for a description of the probes, and instructions on how to design and order them.

xGen® Predesigned Gene Capture Pools

Reaction Size (Rxn) Format Price per Gene Minimum Order
16 rxn Tubes 10 Genes
Plates 10 Genes
96 rxn Tubes 10 Genes
Plates 10 Genes

xGen® Custom Target Capture Probes

Scale Price per Probe Minimum Order
Mini (1 rxn/probe) 200 probes
Standard (8 rxns/probe) 96 probes
XL (64 rxns/probe) 96 probes

$15.00 USD
$25.00 USD
$35.00 USD
$100.00 USD
$100.00 USD
$150.00 USD
$150.00 USD

Customized Capture Panels and Probe Spikes for Existing Panels

xGen® Lockdown® Probes are individually synthesized pools of  5′-biotinylated probes for target capture applications in next generation sequencing. xGen Lockdown Probes are produced using the same industry-leading, high-fidelity synthesis chemistry developed by IDT for our Ultramer® Oligonucleotides.

These probes can be used for creating custom capture panels that can be optimized, expanded, and combined with other panels. xGen Lockdown Probes can also be used to enhance the performance of existing capture panels, rescuing poorly represented regions such as areas of high GC content.

Applications

Create affordable custom capture panels against small target regions:

  • Develop specific sub-panels against target regions of interest
  • Achieve sufficient coverage depth to detect rare alleles
  • Sequence hundreds of samples cost effectively
  • Optimize, expand, or combine custom panels

  Spike into array-derived baits to improve capture performance:

  • Rescue drop-out regions of array-derived exome captures or large captures
  • Improve uniformity of coverage across your target space
  • Discover more genetic mutations with high confidence
  • Selectively improve specific regions of interest without redesigning a new panel


Two Types of Probes for Customized Target Capture Panels

     xGen Predesigned Gene Capture Pools and Plates

  • Human coding regions only
  • More cost effective
  • Use gene symbols or gene IDs (only) to identify targets
  • You must have a minimum of 10 genes/target regions per entry

xGen Predesigned Gene Capture Pools and Plates are already configured probe designs available for all human gene coding sequences. These probes will provide you with customized gene panels most quickly, and at the most cost-effective price. Probe designs are generated using the same design engine used to create the xGen Pan-Cancer Panel, and use 1X tiling with repeat-masking. These probe sets have been demonstrated to provide ~5000-fold enrichment, high coverage over targeted regions, and minimal GC bias (see the Performance Tab).

The xGen Predesigned Gene Capture Pools and Plates are supplied either premixed or with each gene probe pool in a separate plate well, allowing you to vary the set of probe pools used from experiment to experiment. The pools and plates are available in 16 and 96 reaction sets, empowering you to explore different panel compositions to identify the best set of genes for long term projects. To include specific human non-coding sequences in your target capture panel, add xGen Custom Target Capture Probes.

     xGen Custom Target Capture Probes

  • Any species, any sequence
  • Use for human probe targets when Predesigned Gene Capture Pools do not match your criteria for design
  • Transition to high volume applications easily with 3 production scales
  • Use gene symbols, gene IDs, and BED files to identify human, mouse, and rat gene targets; RefSeq accession numbers or FASTA-formatted sequences can also be used
  • Design parameters include target definition, probe length, and tiling density

xGen Custom Target Capture Probesare custom designed probe pools for specialized targeting (e.g., non-coding regions such as promoters and UTRs), and for targeting non-human species. The specialized design and synthesis of these custom probe pools may come at a higher cost compared to xGen Predesigned Gene Capture Pools.

Selecting Your Probes

Use the xGen Lockdown Design Tool (Order Tab) to select target region probe pools for your customized target capture panels. The Design Tool will default to the more economical xGen® Predesigned Gene Capture Pools. Using target input formats other than gene symbols or gene IDs, and/or changing the Custom Design Parameters will allow you to select xGen Custom Target Capture Probes.

You can, of course, create a your own customized targeting panel with a mixture of xGen Predesigned Gene Capture Pools and xGen Custom Target Capture Probes; e.g., create an order for 50 xGen Predesigned Gene Capture Pools, then add a second order for several xGen Custom Target Capture Probes to target UTRs of a subset of those genes.

 

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Protocols

    Product P­rotocols

       PDF, 140.29 KB

      PDF, 136.45 KB

    Additional Reference Protocols

  1. Nijman IJ, Mokry M, et al. (2010) Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples. Nat Methods, 7(11):913–915.A protocol for genomic enrichment of pooled barcoded samples using a single assay that increases experimental flexibility and efficiency. Identified known variants in N-ethyl-N-nitrosourea–mutagenized rats at >96% sensitivity.
  2. Harakalova M, Mokry M, et al. (2011) Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing. Nat Protoc, 6(12):1870–1886. An efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed barcoded samples that is compatible with both microarray- and solution-based capture methods. The protocol enables multiplexing of 3–5 samples for whole-exome experiments and up to 96 samples for targeted sequencing.

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Number of Reactions and Resuspension Volumes

Lockdown® Probes Scale:   
Number of Probes:     probes
Number of Reactions Available:    ###  reactions
Resuspension Volume for 1X:    ### µL

Scale Material Delivered No. of Reactions (Rxn)
 1X Volume
 Mini 3 pmol/probe 1 rxn/probe  1.5 µL/probe
 Standard 24 pmol/probe 8 rxns/probe  12 µL/probe
 XL 192 pmol/probe 64 rxns/probe  96 µL/probe

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Posters, Application Highlights, and Related Articles

Posters

Application Highlights

Additional Related Articles

 

High Target Coverage

xGen Lockdown Probes provide approximately 5000-fold enrichment, high coverage over targeted regions (Figure 1), and minimal GC bias.

Figure 1. xGen® Lockdown® Probes Yield High Coverage. Ligation-based sequencing libraries were amplified by PCR using HiFi DNA polymerase (Kapa Biosystems). A set of 1000 xGen Lockdown Probes spanning the targeted region was used for in-solution hybrid selection with 2 µg library input. Hybridization was performed for 24 hr and enriched targets were sequenced on a HiSeq 2000 System (Illumina) using 49 x 49 paired-end reads. The xGen Lockdown Probes provided greater coverage than array/RNA baits.

Improve Coverage of Array-Synthesized Baits

xGen Lockdown Probes can be added to array-derived RNA baits to enhance the coverage of GC-rich targets, such as first exons (Figure 2).

Figure 2. Spiking In Individually Synthesized Oligonucleotide Baits Improves Sequencing Coverage of Array-Derived RNA Baits.Individually synthesized 5′-biotinylated Ultramer® Oligonucleotides (xGen® Lockdown® Probes) were spiked into commercial, array-synthesized RNA baits to improve coverage of areas with high GC content. Either (A) 1000 Ultramer oligo baits targeting a ~133 kb region, or (B) 3 Ultramer oligo baits targeting a single exon, were mixed with 1.1 MB RNA baits and hybridized for 24 hr, followed by target capture using streptavidin. Enriched targets were sequenced on a HiSeq 2000 system using 49 x 49 paired-end reads. Lockdown probes improved general coverage of GC-rich targets (A)and single GC-rich exons (B).