Multiplexing

PacBio systems provide the ability to sequence multiple samples on a single SMRT Cell to increase throughput and enhance the efficiency of your research.

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Decrease your cost-per-sample with multiplex sequencing

Our multiplexing options allow large numbers of sample libraries to be pooled and sequenced simultaneously on a SMRT Cell. Sample multiplexing is useful when targeting specific genomic regions or working with smaller genomes.

Samples are barcoded by incorporating PacBio-specific barcodes to both ends of the double-stranded DNA so that reads can be identified and sorted before data analysis. Barcodes are located between the hairpin adapters and sample insert.

Multiplexing is supported with three options that provide flexibility to incorporate unique sample identifiers, called barcodes, during target amplification or library preparation. This is achieved through barcoded primers for single-step PCR, tagged barcoded primers via two-step PCR, and barcoded overhang adapters.

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Add barcoded primers to amplicons during (1) PCR through either target-specific primers with incorporated barcodes or (2) a two-step PCR using barcoded universal primers.

Add barcodes to samples with (3) ligation of barcoded SMRTbell adapters to template.

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Setting up multiplexing on Sequel, Sequel II, and IIe systems

Barcoding strategies for multiplexing on PacBio Sequel systems are application dependent. All workflows can include barcodes to uniquely identify samples but differ in how barcodes are incorporated into the SMRTbell templates. List of recommended barcodes, procedures, ordering sheets and FASTA files required for demultiplexing are listed below, by application.

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Whole genome sequencing

As the throughput of PacBio Sequel systems has increased, so has a corresponding growth in multiplexing capacity for whole genome sequencing, specifically for microbial sequencing, de novo assembly and structural variation. Learn more about the whole genome sequencing application and see details on multiplexing below:

Sequel II and IIe systems

Application Available PacBio product Multiplex level FASTA file Plate map file
SMRTbell barcoded adapter plate 3.0 Max = 96
SMRTbell barcoded adapter plate 3.0 Max = 2
Structural variant detection SMRTbell barcoded adapter plate 3.0 Max = 2

Sequel system

Application Available PacBio product Multiplexing level FASTA file Oligo ordering sheet
Barcoded overhang adapter kit 8A and/or Barcoded overhang adapter kit 8B Max = 16 N/A
Multiplexing is not recommended Not recommended N/A N/A
Structural variant
detection
Multiplexing is not recommended Not recommended N/A N/A
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Targeted sequencing

Multiplexing options available for Sequel II systems for targeted sequencing using PCR amplicons or target enrichment with hybridization capture probes. Learn more on our targeted sequencing page.

Sequel II and IIe systems

Application Available PacBio product Multiplexing level FASTA file Oligo order sheet / plate map
Barcoded universal primer Barcoded universal primer plate v2 Max = 96
Barcoded M13 primer plate Max = 384
SMRTbell barcoded adapter plate 3.0 Max = 96
Target enrichment
Oligo synthesis provider Max = 24
Target enrichment with Twist probes Twist Universal Adapter System Max = 384 Download Order from Twist

Sequel system

Application Available PacBio product Multiplexing level FASTA file Oligo ordering sheet
Barcoded universal primer plate v2 Max = 96
Barcoded M13 primer plate Max = 384
Barcoded overhang adapter Kit 8A Barcoded overhang adapter Kit 8B Max = 16
Target enrichment
Oligo synthesis provider Max = 24
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RNA sequencing

The Iso-Seq method is the PacBio solution for sequencing full-length transcripts,the Iso-Seq express workflow is a one-day protocol that requires only 60–300 ng of total RNA and supports multiplexing of different tissues. Learn more about the RNA sequencing application and see details on multiplexing below:

Sequel II and IIe systems

Application Available PacBio product Multiplexing level FASTA file Oligo ordering sheet
Oligo synthesis provider
Max=12

Sequel system

Application Available PacBio product Multiplexing level FASTA file Oligo ordering sheet
Oligo synthesis provider Max=12
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Metagenomics

Multiplex options are available on the PacBio Sequel systems for both full-length 16S sequencing and shotgun metagenomics profiling applications. Learn more about our metagenomic applications for characterizing microbial communities and see details on multiplexing below:

Sequel II and IIe systems

Application Available PacBio product Multiplexing level FASTA file Oligo ordering sheet
Oligo synthesis provider Max = 192
SMRTbell barcoded adapter plate 3.0 Max = 8

Sequel system

Application Available PacBio product Multiplexing level FASTA file Oligo ordering sheet
Oligo synthesis provider Max = 48
Multiplexing is not recommended Not recommended N/A N/A
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SMRT analysis barcode analysis overview and requirements

SMRT Link includes a streamlined end-to-end demultiplex barcodes analysis. Overview and details of available features are described here.

Documentation

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