The Sequel II and IIe Systems are powered by Single Molecule, Real-Time (SMRT) Sequencing, a technology proven to produce highly accurate long reads, known as HiFi reads, for sequencing data you and your customers can trust.
Arabica coffee, revered for its taste and aroma, has a complex genome. It is an allotetraploid (2n=4x=44) with a genome size of approximately 1.3 Gb, derived from the recent (< 0.6 Mya) hybridization of two diploid progenitors (2n=2x=22), C. canephora (710 Mb) and C. eugenioides (670 Mb). Both parental species diverged recently (< 4.2Mya) and their genomes are highly homologous. To facilitate assembly, a dihaploid plant was chosen for sequencing. Initial genome assembly attempts with short read data produced an assembly covering 1,031 Mb of the C. arabica genome with a contig L50 of 9kb. By implementation of long read…
PacBio bioinformatician, Elizabeth Tseng, reviews the bioinformatics strategies utilizing PacBio long-read sequencing data for isoform sequencing for full-length transcript sequencing without assembly.
Genomic instability is one of the hallmarks of cancer, leading to widespread copy number variations, chromosomal fusions, and other structural variations. The breast cancer cell line SK-BR-3 is an important model for HER2+ breast cancers, which are among the most aggressive forms of the disease and affect one in five cases. Through short read sequencing, copy number arrays, and other technologies, the genome of SK-BR-3 is known to be highly rearranged with many copy number variations, including an approximately twenty-fold amplification of the HER2 oncogene. However, these technologies cannot precisely characterize the nature and context of the identified genomic events…
The assembly, annotation, and characterization of the sugar pine (Pinus lambertiana Dougl.) transcriptome represents an opportunity to study the genetic mechanisms underlying resistance to the invasive white pine blister rust (Cronartium ribicola) as well as responses to other abiotic stresses. The assembled transcripts also provide a resource to improve the genome assembly. We selected a diverse set of tissues allowing the first comprehensive evaluation of the sugar pine gene space. We have combined short read sequencing technologies (Illumina MiSeq and HiSeq) with the relatively new Pacific Biosciences Iso-Seq approach. From the 2.5 billion and 1.6 million Illumina and PacBio (46…
Bipolar disorder (BD) is a phenotypically and genetically complex and debilitating neurological disorder that affects 1% of the worldwide population. There is compelling evidence from family, twin and adoption studies supporting the involvement of a genetic predisposition in BD with estimated heritability up to ~ 80%. The risk in first-degree relatives is ten times higher than in the general population. Linkage and association studies have implicated multiple putative chromosomal loci for BP susceptibility, however no disease genes have been identified to date.
The characterization of gene expression profiles via transcriptome sequencing has proven to be an important tool for characterizing how genomic rearrangements in cancer affect the biological pathways involved in cancer progression and treatment response. More recently, better resolution of transcript isoforms has shown that this additional level of information may be useful in stratifying patients into cancer subtypes with different outcomes and responses to treatment.1 The Iso-Seq protocol developed at PacBio is uniquely able to deliver full-length, high-quality cDNA sequences, allowing the unambiguous determination of splice variants, identifying potential biomarkers and yielding new insights into gene fusion events. Recent improvements…
Transcriptome sequencing has proven to be an important tool for understanding the biological changes in cancer genomes including the consequences of structural rearrangements. Short read sequencing has been the method of choice, as the high throughput at low cost allows for transcript quantitation and the detection of even rare transcripts. However, the reads are generally too short to reconstruct complete isoforms. Conversely, long-read approaches can provide unambiguous full-length isoforms, but lower throughput has complicated quantitation and high RNA input requirements has made working with cancer samples challenging. Recently, the COLO 829 cell line was sequenced to 50-fold coverage with PacBio…
In this ASHG workshop presentation , Jonas Korlach, CSO of PacBio, walked attendees through recent product updates and the coming technology roadmap. The Sequel System 6.0 release offered major improvements to accuracy, throughput, structural variant calling, and large-insert libraries, he said, showing examples of 35 kb libraries. Looking ahead, Korlach said that the V2 express library preparation product should be available early in 2019, with the new 8M SMRT Cell being introduced sometime later.
In this ASHG workshop presentation, Elizabeth Tseng of PacBio showed how the Iso-Seq method can be used to discover disease-associated alternative splicing. Because this approach to isoform sequencing yields accurate, full-length transcripts requiring no assembly, it’s ideal for disease studies that need a more comprehensive picture of alternative splicing activity. Tseng offered several published examples of how the Iso-Seq method has been used for everything from single-gene studies to whole-transcriptome studies, and also detailed how the latest Sequel System chemistry recovers more genes and produces more usable reads.
In this PacBio User Group Meeting presentation, PacBio scientist Kristin Mars speaks about recent updates, such as the single-day library prep that’s now possible with the Iso-Seq Express workflow. She also notes that one SMRT Cell 8M is sufficient for most Iso-Seq experiments for whole transcriptome sequencing at an affordable price.
In this PacBio User Group Meeting presentation, Shawn Trojahn of Washington State University describes transcriptome sequencing and analysis of grizzly bears focused on differential gene expression during hibernation and active cycles, potentially offering human-relevant information about muscle atrophy and insulin resistance. The team was able to identify more unique isoforms just from liver tissue than had been previously characterized in the entire reference genome. Of particular interest: more than 2,000 transcripts differentially expressed between hibernation and active season, including 86 genes that have isoforms expressed in opposite directions.
In a push to develop insect-based food sources for people, Brenda Oppert from the USDA has been sequencing bug genomes with PacBio technology. Long reads are essential because of the highly repetitive sequences and large genomes. On the Sequel II System, a single SMRT Cell is sufficient to generate 350-fold coverage and produce a high-quality assembly for some of the insects she’s studying.
During a PAG 2020 workshop, Zev Kronenberg, a senior bioinformatics engineer at PacBio, describes how he used the PacBio Iso-Seq transcriptome sequencing and analysis method to annotate great ape genomes, detangle several complicated loci, and enrich our biological understanding of the differences between apes and humans.
In this webinar, Adam Ameur of SciLifeLab at Uppsala University shares how he uses Single Molecule, Real-Time (SMRT) Sequencing applications for medical diagnostics and human genetics research, including sequencing of single genes and de novo assembly of human genomes as well as a new method for detection of CRISPR-Cas9 off-targets.