Multiplex Sequencing

Processing more samples in less time

Increase the number of samples sequenced per run and optimize high-throughput sequencing

Sample Multiplexing Overview

Sample multiplexing, also known as multiplex sequencing, allows large numbers of libraries to be pooled and sequenced simultaneously during a single run on Illumina instruments. Sample multiplexing is useful when targeting specific genomic regions or working with smaller genomes. Pooling samples exponentially increases the number of samples analyzed in a single run, without drastically increasing cost or time.

With multiplex sequencing, individual "barcode" sequences are added to each DNA fragment during next-generation sequencing (NGS) library preparation so that each read can be identified and sorted before the final data analysis. These barcodes, or index adapters, can follow one of two major indexing strategies depending on your library prep kit and application.

Conceptual Overview of Sample Multiplexing

Multiplex Sequencing Highlights

  • Fast high-throughput strategy: Large sample numbers can be simultaneously sequenced during a single experiment
  • Cost-effective method: Sample pooling improves productivity by reducing time and reagent use
  • Simplified analysis: Automatic sample identification with "barcodes" using Illumina data analysis software
Sample Multiplexing Facilitates Microbial Studies

Researchers leverage the speed and multiplex sequencing capabilities of Illumina sequencers to identify microbes along the Mississippi River.

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Related Topics in Multiplex Sequencing

Dual Index Sequencing

When preparing libraries for multiplexing, we recommend using unique dual indexes. Unique dual indexes allow you to increase the number of samples sequenced per run and reduce per-sample cost compared to other indexing strategies.

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Unique Molecular Identifiers

Unique molecular identifiers (UMIs) are a type of molecular barcoding that provides error correction and increased accuracy during sequencing. Sequencing with UMIs can reduce the rate of false-positive variant calls and increase sensitivity of variant detection.

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Minimizing Index Hopping

With multiplexing, the potential for index hopping is present regardless of the library prep method or sequencing system used. Learn best practices to help minimize the effects of index hopping.

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Chan Zuckerberg Biohub and the NovaSeq System

Researchers at the Chan Zuckerberg Biohub are working to conduct innovative experiments and form new collaborations in genomics. The NovaSeq System enables them to bundle many samples together for medical research that may help to improve human health.

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Related Content

  • High-Throughput Sequencing Methods: Cost-effectively run emerging data-rich methods, including single-cell and spatial analyses, and process more samples to improve statistical power.
  • Library Prep Automation: Learn about automated liquid handling solutions designed to help labs prepare large quantities of sequencing libraries.
  • Pooling Calculator: Dilute pooled libraries to the appropriate concentration for multiplex sequencing.
Flexible, Scalable Sequencer

The NovaSeq 6000 System offers scalable throughput and flexibility for virtually any genome, sequencing method, and scale of project.

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