Next Generation Sequencing
Support and Educational Content

A solution for sample cross-talk and index hopping in multiplexed NGS

Unique, dual-matched adapters substantially reduce cross-talk errors

For most applications, the massive sequencing capabilities of current NGS platforms is far greater than the sequencing requirements for any single sample. Therefore, multiplexing several samples in a high-throughput sequencing run is an indispensable technique to maximize use of instrument capacity.

For multiplexing to be successful, artificial, sample-specific, index sequences are added during library preparation. After sequencing, during a process called demultiplexing, the indices are used to assign the millions of individual reads to the correct sample.

Historically, a relatively small number of unique sequences (8 x i5 and 12 x i7) have been used to create index combinations to identify multiplexed samples. Unfortunately, various mechanisms can cause the wrong index to be attached to some of the sequences from a sample. This physical addition of an index to the wrong sample is referred to as “index hopping”. With the combinatorial approach, a single index swap may cause a read to be mis-assigned to a different sample causing cross-talk.

In the webinar, Unique, dual-matched adapters mitigate index hopping between NGS samples, Dr Kristina Giorda explains the sources of sample cross-talk and discusses how unique, dual-matched adapters can facilitate the removal of errors from NGS data analysis. Throughout the presentation, Dr Giorda also offers advice for library preparation and target enrichment that can help reduce the chances of index hopping that contribute to sample cross-talk. Watch the webinar recording below to learn more about how to reduce sample cross-talk in your sequencing experiments.

For more information on these xGen® Dual Index UMI Adapters, or to place customized orders, please contact our scientific applications support group.

Product focus: Target capture reagents

xGen® Dual Index UMI Adapters

For more information on these xGen® Dual Index UMI Adapters, or to place customized orders, please contact our scientific applications support group.

xGen Lockdown® Probes—target capture probe pools for NGS

xGen Lockdown Probes are pools of individually synthesized, quality-controlled, and normalized hybridization probes. Use them to generate custom capture panels for targeted sequencing or to enhance the performance of existing panels. xGen Predesigned Gene Capture Probe Pools are available for any human RefSeq coding gene. Select from predesigned and custom probes that offer:

  • Sensitive detection of SNPs, indels, CNV, LOH, and translocations
  • GMP compliance for clinical and diagnostics research
  • Flexibility to augment existing panels or create completely custom panels
  • Quick delivery

Discover more about xGen Lockdown Probes.

xGen Lockdown Panels

xGen Lockdown Panels are preconfigured, validated, and stocked pools of xGen Lockdown Probes for targeted next generation sequencing of defined gene families:

  • xGen Exome Research Panel
  • xGen Acute Myeloid Leukemia Panel
  • xGen Pan-Cancer Panel
  • xGen Inherited Diseases Panel
  • xGen Human ID Research Panel
  • xGen Human mtDNA Research Panel

Discover more about xGen Lockdown Panels.

xGen Lockdown Reagents—hybridization and wash kit

Achieve uniform coverage with hybridization and wash buffers that are optimized for target enrichment using xGen Lockdown Probes and Panels. A short, 4-hour hybridization protocol generates results quickly.

Discover more about xGen Lockdown Reagents.

xGen Blocking Oligos

xGen Blocking Oligos prevent adapter cross hybridization and minimize off-target capture, increasing specificity during targeted sequencing. Universal Blocking Oligos—TS Mix are designed for use with Illumina barcoded adapters. This ready-to-use mix effectively blocks dual- or single-index adapters to significantly improve on-target performance.

Also available are:

  • xGen Standard Blocking Oligos
  • xGen Custom Blocking Oligos
  • xGen Blocking Oligos for diagnostics—contact for information
  • xGen Blocking Oligos for Ion Torrent adapters

Discover more about xGen Blocking Oligos.

Additional NGS webinars and articles

Analyzing the exome—focus your NGS analysis with high-performance target capture—Webinar summary: Targeted sequencing capture, using probe pools or panels, can increase read depth and the number of samples per run, while decreasing sequencing cost and simplifying data analysis. See how using individually synthesized, quality checked, DNA target capture probes (xGen® Lockdown® Probes) covering the human exome (xGen Exome Research Panel) perform across a variety of metrics and compare to other available exome panels.

NGS detection of low frequency genetic variants using novel, molecular sequencing adapters—Webinar summary: Watch this webinar recording to learn about unique molecular adaptors and a high-performance target capture method for NGS analysis of low frequency variants.

NGS target capture recommendations for FFPE samples—Webinar summary: Learn how it is possible to create high quality target capture libraries from formalin-fixed, paraffin-embedded samples. Dr Kristina Giorda presents an FFPE sample workflow with a concise explanation of DNA quality analysis and how quality assessment can be used to guide the amount of DNA input for NGS library preparation.

Get the most out of your NGS samples—expandable tumor and disease target capture panels—Learn about expandable NGS target capture panels that enrich for mutated genes implicated in tumors and genes associated with inherited diseases. Designed in collaboration with experts from the Emory Genetics Laboratory and the Cancer Genome Atlas, both panels return consistent results with high reproducibility and deep uniform coverage.

Review other DECODED Online newsletter articles on NGS applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Hans Packer, PhD, is a scientific writer at IDT.

© 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see

xGen® Blocking Oligos

Adapter blocking oligos increase the number of on-target reads by preventing non-specific binding during hybridization.

Find blockers for your platform ≫

Related Articles

Target Enrichment Facilitates Focused Next Generation Sequencing

The rationale and benefits of enriching subsets of the genome (target enrichment by hybrid capture) prior to sequencing.

Read more ≫

Improving Uniform Coverage of Targeted Sequences for NGS

The challenges faced in obtaining uniform coverage of NGS data and how IDT xGen® Lockdown® Probes are uniquely positioned to facilitate uniform sequence coverage.

Read more ≫

Towards Providing Personalized Medicine—Considerations for Reliable NGS Data

Geneseeq Technology, Inc. demonstrates how they improved their target capture methods to increase accuracy in clinical diagnostics by using optimized blocking oligos and stringent hybridization conditions.

Read more ≫

Delivering Comprehensive Genomic Profiling for Clinical Cancer Care Using Targeted Sequencing

Read how scientists at Foundation Medicine, Inc use hybrid selection in their FoundationOne® Test to help clinicians select patient specific treatment options.

Read more ≫

Insertion Site Detection and Targeted RNA Capture Using Next Generation Sequencing

Scientists at Cofactor Genomics use in-solution hybridization to focus on regions of interest for next generation sequencing.

Read more ≫