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Decrease your cost-per-sample with multiplex sequencing

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

Multiplex sequencing allows 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.

Barcode Detection

Multiplexing is supported with three barcoding options that provide flexibility to incorporate unique sample identifiers 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.

See Figures

Setting Up Multiplexing on Sequel, Sequel II and IIe Systems

Barcoding strategies for multiplex sequencing 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.


Whole Genome Sequencing

As the throughput of PacBio Sequel Systems has increased, there has been a corresponding growth in multiplexing capacity for whole genome sequencing, specifically for microbial sequencing, de novo assembly and structural variation. See details below:

Sequel II and IIe Systems

Sequel System

RNA Sequencing

The Iso-Seq Express workflow is a 1-day protocol that requires only 60-300 ng of total RNA and supports multiplexing of different tissues. Specific details below:

Sequel II and IIe Systems

Sequel System

Metagenomics

Multiplex sequencing is available on the PacBio Sequel Systems for both Full-length 16s sequencing and Shotgun metagenomics profiling. Details below:

Sequel II and IIe Systems

Sequel System

Targeted Sequencing

Depending on the amplicon size, multiplex up to 384 barcodes for targeted sequencing. See details below:

Sequel II and IIe Systems

Sequel System


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.

Contact us to learn more about how this innovative software can support your research efforts.

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