Sequencing is the backbone of modern genomics. It has evolved over time, and as platforms grow to be faster and more accurate, researchers are finding new ways to maximize every run. One approach – multiplexing – allows multiple samples to be processed in parallel, increasing efficiency, lowering per-sample costs, and expanding the scope of experiments.
Here, we’ll look at the principles behind multiplexing in sequencing, how it’s implemented on PacBio HiFi systems, and where it delivers the most impact, from RNA analysis to targeted and low-input applications.
What is multiplexing and why is it used in sequencing?
Multiplexing refers to the process of combining multiple samples into a single sequencing run. Instead of preparing and sequencing each sample individually, researchers use barcodes, unique DNA sequences added to each sample’s library, to keep them distinguishable within the pooled mixture. After sequencing, the reads can be sorted back into their original sample groups in a step called demultiplexing.
The motivation for multiplexing is straight forward: today’s sequencing platforms generate more data than many individual samples require. Without multiplexing, that excess capacity goes to waste or requires researchers to artificially bulk up each library with more DNA, which isn’t always practical or even possible.
By enabling multiple samples to share the sequencing capacity of a single run, multiplexing allows scientists to:
- Reduce costs by dividing sequencing expenses across multiple samples.
- Increase workflow efficiency by preparing and processing samples in parallel.
- Minimize confounding variables by sequencing samples together, reducing batch effects and improving reproducibility.
How does multiplexing work?
The general principles of multiplexing include:
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- Barcoding: During library preparation, each sample is ligated or otherwise tagged with a unique barcode sequence, or index, a short, identifiable stretch of DNA that differentiates one sample from another (Figure 1).
- Pooling: Barcoded libraries are then pooled into a single mixture. The pool is loaded onto the sequencer, which treats it as a single library.
- Sequencing: The sequencer reads both the barcode and the target DNA fragments during the sequencing run.
- Demultiplexing: After sequencing, bioinformatic tools identify the barcodes associated with each read and assign it back to the appropriate sample. This process effectively groups the pooled data into individual datasets.
Figure 1: The SMRTbell hairpin and 10 bp index sequence flanking a DNA insert.
It is very important that the barcoding and demultiplexing processes are highly accurate. Misassigned reads could lead to contamination across samples and compromise downstream analyses.
Key considerations when multiplexing samples
While multiplexing offers significant advantages, successful implementation requires careful planning and execution. Here are several important factors to keep in mind:
Barcode compatibility and design
Barcodes must be easily distinguishable from one another, even in the presence of sequencing errors. This often involves designing barcode sets that are balanced in terms of GC content and spaced sufficiently apart in sequence similarity to avoid misclassification.
Uniformity of sample representation
A major performance metric in multiplexed sequencing is pooling uniformity. This represents how evenly data is distributed across samples in the pool. Uniformity is typically measured using the coefficient of variation (CV):
CV = (standard deviation / mean) of the data yield across samples.
Low CV values indicate high uniformity, meaning each sample receives a similar number of reads. This is critical when comparing data between samples, such as in differential expression or variant analysis studies.
Experimental design and downstream analysis
Multiplexing can improve experimental consistency by allowing multiple related samples to be processed and sequenced together. However, it also introduces complexity during the data analysis stage, especially when sample numbers are high. Clear labeling, barcode tracking, and robust demultiplexing tools are essential.
How multiplexing works for PacBio HiFi sequencing
All PacBio HiFi sequencing platforms, including Revio and Vega systems, support robust and efficient multiplexing workflows that allow for the pooling of multiples samples on a single SMRT Cell to share the capacity and split the cost of sequencing.
Barcoding and pooling with PacBio
For HiFi sequencing, PacBio offers multiple options for barcoding, each tailored to different sample types and workflows:
- SMRTbell adapter indexes: 384 unique barcodes designed to be ligated to SMRTbell libraries.
- Kinnex adapter indexes: Used for RNA sequencing workflows, including bulk and single-cell RNA-seq.
- Barcoded M13 primers: Enable barcoding at the PCR stage, useful for targeted amplification strategies.
- Twist Bioscience UDI adapters: Compatible with PacBio workflows like Ampli-Fi, these adapters are used during whole-genome amplification (WGA).
After barcoded libraries are pooled and sequenced, demultiplexing occurs either on instrument (on the Revio and Vega systems for SMRTbell and Kinnex adapters) or post-run in SMRT Link software. The output for on-instrument demultiplexing is a separate HiFi BAM file for each sample, ready for downstream analysis.
Applications of multiplexing in HiFi sequencing
Multiplexing makes sequencing more cost-effective and enables powerful applications that would otherwise be logistically or financially impractical. Several PacBio workflows are optimized specifically to take advantage of multiplexing:
Kinnex RNA sequencing
Kinnex RNA sequencing offers full-length isoform information with full-length isoform discovery and abundance information, cell-type specific isoform discovery, and species-level metagenomics community identification that are difficult to capture with short reads. RNA samples are barcoded during cDNA synthesis, offering sample-level multiplexing. All Kinnex kits utilize the same set of 4 barcoded adapters to enable library-level multiplexing. With Kinnex multiplexing, researchers can process hundreds of RNA samples together, reducing costs and improving reproducibility.
PureTarget for repeat expansions and carrier screening
PureTarget leverages a CRISPR-Cas9 system to target challenging, clinically-relevant regions with an amplification-free approach. Because PureTarget is often performed across many samples, multiplexing is essential to make the approach scalable and cost-effective.
Figure 3: Targeting is done using Cas9 and a pair of guide RNAs flanking the region of interest. Samples are barcoded by ligating indexed SMRTbell adapters during library prep. Barcodes are shown in dark blue. Nuclease treatment removes non-SMRTbell templates prior to sample pooling and sequencing.
Ampli-Fi for ultra-low input DNA
Ampli-Fi is a protocol that enables HiFi sequencing for samples with limited DNA input, down to just 1 ng. Given the precious nature of these samples, multiplexing becomes especially valuable, because it allows researchers to maximize data from each sequencing run, while still recovering accurate, haplotype-resolved genome data for each sample.
Multiplexing with Twist UDIs can be performed either just after amplification to save on cost and time, or alternatively following the Ampli-Fi protocol just before sequencing.
High-throughput sequencing leads the way for multiplexing
Large transcriptomic projects, ultra-low input sequencing, and population-scale variant studies all rely on multiplexing as a core technique. It makes each run more efficient, cutting costs and turnaround times while giving researchers greater control over their experiments.
On PacBio HiFi systems, multiplexing pairs high-accuracy long reads with flexible barcoding and simple demultiplexing, enabling high-throughput work without sacrificing quality. As long-read sequencing capacity expands, it will remain a key part of turning DNA and RNA data into meaningful insights.