We are upgrading your website experience! Click here for more information.

Next Gen Sequencing

Protocols and Support

Introduction to IDT's XGen® Lockdown® Design Tool for Custom NGS Target Capture

This video provides a quick introduction to the design of custom target enrichment probes using IDT's xGen Lockdown Design Tool, which is available on the web free of charge (IDT account required.)

Additional Resources

Related Articles

How Does IDT Evaluate Its Target Capture Panels?

How Important Are Those NGS Metrics?

NEW! Blocking Oligos for NGS Target Enrichment Applications

Next Generation Sequencing in the Clinic: A Perspective from Dr Elaine Mardis

Reduced Barcode Contamination using Oligonucleotides with TruGrade™ Processing

xGen® Lockdown® Probes

Custom Target Capture Probes for NGS Target Capture


Benefits

  • Achieve higher performance with capture probes that are individually assessed for QC
  • Start your project faster with a 7–10 day turnaround time
  • Develop custom panels by pooling up to 2000 probes per tube
  • Transition to high volume applications easily with 3 production scales

Features

  • Probes are individually assessed by mass spectrometry for QC
  • All probes contain a 5' biotin modification
  • Maximum of 2000 probes per pool
  • Probes are shipped in 7–10 days


xGen® Lockdown® Probes

Use our xGen Lockdown design tool to generate custom capture panels for enrichment of your targets of interest. We currently accept gene IDs, gene symbols and chromosomal coordinates for human, mouse and rat gene targets as well as RefSeq accessions and FASTA-formatted sequences. Design parameters include probe size, tiling depth and repeat masking.

Scale (Rxns/Probe)
Price per Probe
Minimum Order
Mini (1 rxn/probe)
Standard (8 rxns/probe)
XL (64 rxns/probe)
$15.00 USD
$25.00 USD
$35.00 USD
200 probes
96 probes
96 probes

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 of 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 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).

Heading

Designing Your Probes

Overview

IDT currently accepts Gene ID, Gene Name, and FASTA as target definition inputs for standard designs. Design parameters include probe size, tiling depth, maximum flank size, and repeat masking. By default, we allow probes to overlap masked target regions by up to 20 bases. Other inputs and parameters are accepted, but may require additional fees.

The fee structure for custom designs is as follows:

Time Cost
First 2 Hours Free
Additional $50/Hour

Up to $250 worth of design fees can be credited towards your corresponding xGen Lockdown Probe order.

If your targets are documented by chromosome location, please follow the instructions below to obtain acceptable FASTA sequences for design requests from the UCSC genome browser.

Extracting Sequence Information from the UCSC Genome Browser

Note: These instructions assume that you have a bed file with genomic coordinates for each region.

  1. Make sure you have a correctly formatted bed file with genomic coordinates. UCSC accepts bed files with 4 columns: the first column is the chromosome number; the second column is the genomic coordinate that specifies the beginning of your sequence; the third column is another genomic coordinate that specifies the end of your sequence; and the fourth column is the name of the location, which you can define. There are no headers in bed files. bed files can be opened with a text editor such as Notepad.
  2. Go to the UCSC genome browser home page. Click on the Genomes tab at the top of the page.
  3. Select the genome and assembly you want to use as the reference for your sequence extraction in the drop boxes on the top of the page.
  4. Click on the manage custom tracks button.
  5. Click add custom tracks.
  6. Click Choose File above the Paste URLs or data box and choose the bed file you want to use or paste your bed file into that box.
  7. Click Submit.
  8. There should be a new track visible in the table at the top of the next page that will include the number of distinct regions in your bed file. Click on the go to table browser button next to this table.
  9. For output file, specify a name for your output file.
  10. For file type returned, select plain text. If you expect your file to be very large and you can decompress .gz files, select the gzip compressed option.
  11. Click get output.
  12. Specify how many flanking bases you want incorporated into your design upstream and downstream of your targets. IDT recommends at least 20 bases.
  13. Under Sequence Formatting Options, select All upper case.
  14. If repeat masking is desired, check the Mask repeats box and select to lower case.
  15. Click get sequence.
  16. Retrieve and review your file.

Heading

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  1X Volume
 Mini 3 pmole/probe 1 reaction/probe  1.5 µL/probe
 Standard 24 pmole/probe 8 reactions/probe  12 µL/probe
 XL 192 pmole/probe 64 reactions/probe  96 µL/probe

Heading

Documents and Materials

Heading

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.