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PacBio sequencing

The accurate long-read Next-Generation Technology

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Overview

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Workflow

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Applications

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ACHIEVE THE MOST DETAILED SEQUENCING BY COMBINING PACBIO'S HIFI APPROACH WITH SEQUEGENICS POWERFUL PROPRIETARY TOOLS FOR MULTIPLE APPLICATIONS:

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  • Metagenomics

  • Viral quasispecies

  • Gene panels

  • de novo genome assembly

  • Large genomic rearrangements

  • Haplotype phasing

  • RNAseq/Isoform

  • Targeted sequencing

Metagenomics

Exploring the diversity of microbial communities is simplified by using a long-read NGS approach. The length of the sequencing reads, combined with the accuracy achieved by PacBio's HiFi approach facilitates straightforward identification of bacterial species that coexist in a single sample, such as soil. While searching for specific sequence motifs is simplified using HiFi, differentiating closely related species can become challenging. Implement Sequegenics' proprietary tools to improve resolution for this and other particularly difficult tasks.

Study of viral quasispecies

Just like studying microbial diversity, implement long-read NGS to perform a detailed characterization of viral quasispecies. The length of the sequencing reads are able to span most (if not all) the genome of virtually any virus, and the haplotype resolution achieved with highly accurate long reads can produce valuable insights into the dynamics of viruses within the host. A combination of PCR-based sample preparation with PacBio's HiFi approach and Sequegenics proprietary tools can produced the most accurate and detailed characterization of viral quasispecies.

Gene panels

For certain conditions, the study of a specific sets of genes can provided critical information for diagnosis and treatment. While gene panel tests are widespread used in clinical genomics, technical limitations of SNP arrays or short-read NGS can cloud the interpretation of the results. Particularly critical for this type of tests is the ability to differentiate active genes from pseudogenes to correctly assign mutations to one or the other. The length of the sequencing reads produced by long-read NGS are able to capture the necessary genetic information to accurately facilitate such distinction, therefore helping producing a much clearer picture of the genetic makeup of the genes targeted.

de novo assembly

Long-read NGS facilitates building genome assemblies that do not require a reference sequence. This approach prevents possible bias introduced by the reference sequence making long-read NGS particularly useful to sequence new species, and drive more diverse human genome databases. Preventing reference bias is particularly relevant for clinical genomics. At Sequegenics, we are currently involved in projects aimed at better understanding the genetic architecture of disease among different ethnicities. Combining de novo assembly facilitated by long-read NGS with Sequegenics proprietary tools for variant calling produce the most accurate genomes assemblies.

Detection of large genomic rearrangements

Genomic rearrangements such as inversions, duplications, and translocations can involve segments of thousands of nucleotides making them particularly challenging to detect with short-read NGS. This is the case for large deletions and insertions as well. Implementing long-read NGS is the best approach to detect this type of structural variants and in PacBio sequencing, limited sequencing coverage can be enough to identify this type of variation, making the study of genomic rearrangements one of the most cost-effective PacBio implementations. At Sequegenics' cloud platform, preset pipelines for SV discovery are available which let users identify this complex type of variation by simply uploading the sequencing output produced by the instrument.

Haplotype phasing

Resolving the cis/trans location of polymorphisms across the genome can be a valuable piece of information for understanding disease and improve diagnostics. From highly conserved to highly variable genes, how mutations are distributed among different alleles can add a level of sequencing resolution that is only possible to achieve with long-read NGS. By leveraging the accuracy of PacBio's HiFi approach, resolving haplotype configurations can become quite simple. In addition, Sequegenics' proprietary tools for haplotype calling can facilitate long range haplotype phasing using both CCS and raw sequencing reads. 

RNAseq - Full transcripts and isoform characterization

Alternative splicing is a fascinating mechanism by which the same precursor mRNA can lead to multiple mRNAs that combine different segments of the original precursor. Thanks to this mechanism, the same gene can produce multiple different products. Differentiating multiple transcripts or isoforms that are the product of the same gene can be extremely challenging for short-read NGS because the difference between them does not relay on mutations but in length and combination of segments of the original precursor. With an average size of 2,000 nucleotides, mRNAs are a perfect target for PacBio's HiFi approach, facilitating multiples passes of the polymerase on every retrotranscribed dsDNA, and producing significant CCS yield.

Targeted sequencing

Sequencing relatively small segments of DNA such as individual genes has become a very popular implementation for long-read NGS, because the current sequencing yield helps reducing sequencing costs of such targets quite dramatically through massive multiplexing. In particular, the high accuracy of PacBio CCS reads is making downstream analytics quite straightforward. Laboratories that need to sequence hundreds of samples can entirely replace Sanger sequencing for a cost-effective long-read approach. HLA sequencing is the most popular example among a diverse list of targets for this technology.

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