PacBio SMRT sequencing

PacBio SMRT sequencing generates reads tens of kilobases in length enabling high quality genome assembly, structural variant analysis, amplicon resequencing, full-length transcript isoform sequencing, full-length 16S rRNA sequencing and amplification free epigenetic characterization.

PacBio Revio utilizes the Single Molecule Real Time Sequencing (SMRT) technology which provides high sensitivity, high throughput and fast turnaround times. It is suitable for all types of de novo genome sequencing, analysis of structural variation and full-length transcript isoform sequencing. Other typical applications include meta-barcode studies and pooled amplicons, as sequencing bias toward shorter fragments is low. Since no amplification is required prior to sequencing, methylations in genomic DNA can be detected by analysing interpulse duration times.

Sample requirements

• Good quality high molecular weight DNA.
• Genomic: > 5–10 µg of DNA per sample and SMRTcell.
• Amplicon: > 0.5–2 µg per sample depending on amplicon length and level of multiplexing.
• Iso-Seq: > 0.5 µg of total RNA with a concentration of least 45 ng/µl and a RIN value 8–10 is required.

There are options of using less DNA for constructing genomic libraries as well, but those might not be applicable to all of the projects. Please contact NGI for more details.

DNA sequencing

With the capacity to generate long reads of very high accuracy it is suitable for all types of de novo genome sequencing, analysis of structural variation and amplicon-based sequencing.

de novo assembly

PacBio de novo genome assemblies deliver megabase-size contig N50s, consensus accuracies >99.99%, and phased haplotypes so you can generate reference-quality assemblies of diverse species and populations. Whole genome sequencing for de novo assembly enables to characterize the complete genetic diversity within a species, to develop population-specific reference genomes to drive precision medicine and assemble microbial genomes and their plasmids in a single experiment. We have experience sequencing a vast range of organisms and genome sizes and can help with everything from DNA extraction to genome assembly.

Genome resequencing

PacBio can be used for whole genome resequencing for phasing and variant detection. The long sequence reads enable characterization of complex structural variants also in complex regions of the genome.

Targeted resequencing (amplicon sequencing)

Amplicon sequencing involves sequencing of PCR-amplified loci across large sets of samples. PacBio can be used to generate highly accurate sequences from amplicons ranging in size from several hundred bases to 10 kb or larger.

Environmental sequencing

PacBio sequencing allows for the identification of individual members of complex microbial populations, with 16S rRNA species-level identification and complete plasmids or viral quasispecies. This technology can resolve complex populations in viral or metagenomic samples, map order and orientation of multiple-inserted phage element, facilitate study of viral integration and integration sites, perform multiplexed multi-locus sequence typing (MLST).

RNA sequencing

Single Molecule, Real-Time (SMRT) Sequencing and Iso-Seq analysis allow you to generate full-length cDNA sequences, with no assembly required. This makes it an ideal method to characterize transcript isoforms within targeted genes or across an entire transcriptome. PacBio Iso-Seq analysis enables discovery of new genes, transcripts and alternative splicing events. It will help improve genome annotation to identify gene structure, regulatory elements and coding regions. The iso-form level resolution may also be used to increase accuracy of RNA-seq quantification generated from short-read data.

Read more about RNA sequencing: Human applications or Plants and animals.

Epigenetics

Single Molecule, Real-Time (SMRT) Sequencing is amplification free and can therefore directly detect epigenetic modifications by measuring kinetic variation during base incorporation. Modification data is captured simultaneously with sequence data, thus eliminating the need for special sample preparation and additional sequencing. Deep coverage is required to resolve the modification signals and it is therefore primarily recommended for microbial genomes. The m6A and m4C methylation status can be determined at each position using this method.

Single Molecule, Real-Time (SMRT) Sequencing is amplification free and can therefore directly detect epigenetic modifications by measuring kinetic variation during base incorporation. Modification data is captured simultaneously with sequence data, thus eliminating the need for special sample preparation and additional sequencing. Deep coverage is required to resolve the modification signals and it is therefore primarily recommended for microbial genomes. The m6A and m4C methylation status can be determined at each position using this method.

User-prepared libraries

NGI accepts user-prepared libraries for PacBio sequencing. Please contact a project coordinator for further information.

Last Updated: 5th October 2024