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June 1, 2021  |  

Full-length transcript profiling with the Iso-Seq method for improved genome annotations

Incomplete annotation of genomes represents a major impediment to understanding biological processes, functional differences between species, and evolutionary mechanisms. Often, genes that are large, embedded within duplicated genomic regions, or associated with repeats are difficult to study by short-read expression profiling and assembly. In addition, most genes in eukaryotic organisms produce alternatively spliced isoforms, broadening the diversity of proteins encoded by the genome, which are difficult to resolve with short-read methods. Short-read RNA sequencing (RNA-seq) works by physically shearing transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest. In contrast, Single Molecule, Real-Time (SMRT) Sequencing directly sequences full-length transcripts without the need for assembly and imputation. Here we apply the Iso-Seq method (long-read RNA sequencing) to detect full-length isoforms and the new IsoPhase algorithm to retrieve allele-specific isoform information for two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata).


June 1, 2021  |  

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be economical for obtaining sequence information for defined regions of the genome. However, most target enrichment methods are reliant upon some form of amplification which can negatively impact downstream analysis. For example, amplification removes epigenetic marks present in native DNA, including nucleotide methylation, which are hypothesized to contribute to disease mechanisms in some disorders. In addition, some genomic regions known to be causative of many genetic disorders have extreme GC content and/or repetitive sequences that tend to be recalcitrant to faithful amplification. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system to target individual genes. This method, in conjunction with the long reads, high consensus accuracy, and uniform coverage of SMRT Sequencing, allows accurate sequence analysis of complex genomic regions that cannot be investigated with other technologies. Using this strategy, we have successfully targeted a number of repeat expansion disorder loci (HTT, FMR1, ATXN10, C9orf72).With this data, we demonstrate the ability to isolate thousands of individual on-target molecules and, using the Sequel System, accurately sequence through long repeats regardless of the extreme GC-content. The method is compatible with multiplexing of multiple target loci and multiple samples in a single reaction. Furthermore, because there is no amplification step, this technique also preserves native DNA molecules for sequencing, allowing for the direct detection and characterization of epigenetic signatures. To this end, we demonstrate the detection of 5-mC in the CGG repeat of the FMR1 gene that is responsible for Fragile X syndrome.


June 1, 2021  |  

Full-Length RNA-seq of Alzheimer brain on the PacBio Sequel II System

The PacBio Iso-Seq method produces high-quality, full-length transcripts and can characterize a whole transcriptome with a single SMRT Cell 8M. We sequenced an Alzheimer whole brain sample on a single SMRT Cell 8M on the Sequel II System. Using the Iso-Seq bioinformatics pipeline followed by SQANTI2 analysis, we detected 162,290 transcripts for 17,670 genes up to 14 kb in length. More than 60% of the transcripts are novel isoforms, the vast majority of which have supporting cage peak data and polyadenylation signals, demonstrating the utility of long-read sequencing for human disease research.


June 1, 2021  |  

Structural variant in the RNA Binding Motif Protein, X-Linked 2 (RBMX2) gene found to be linked to bipolar disorder

Bipolar disorder (BD) is a phenotypically and genetically complex 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 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 BD susceptibility, however no disease genes have yet to be identified. Here, we have fully characterized a ~12 Mb significantly linked (lod score=3.54) genomic region on chromosome Xq24-q27 in an extended family from a genetic isolate that was using long-read single molecule, real-time (SMRT) sequencing. The family segregates BD in at least 4 generations with 16 individuals out of 61 affected. Thus, this family portrays a highly elevated reoccurrence risk compared to the general population. It is expected that the genetic complexity would be reduced in isolated populations, even in genetically complex disorders such as BD, as in the case of this extended family. We selected 16 key individuals from the X-chromosomally linked family to be sequenced. These selected individuals either carried the disease haplotype, were non-carriers of the disease haplotype, or served as married-in controls. We designed a Nimblegen capture array enriching for 5-9 kb fragments spanning the entire 12 Mb region that were then sequenced using long-read SMRT sequencing to screen for causative structural variants (SVs) explaining the increased risk for BD in this extended family. Altogether, 192 SVs were detected in the critically linked region however most of these represented common variants that could be seen across many of the family members regardless of the disease status. One SV stood out that showed perfect segregation among all affected individuals that were carriers of the disease haplotype. This was a 330bp Alu deletion in intron 4 of the RNA Binding Motif Protein, X-Linked 2 (RBMX2) gene that has previously been shown to play a central role in brain development and function. Moreover, Alu elements in general have also previously been associated with at least 37 neurological and neurodegenerative disorders. In order to validate the finding and the functionality of the identified SV further studies like isoform characterization are warranted.


June 1, 2021  |  

A complete solution for high-quality genome annotation using the PacBio Iso-Seq method

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. We describe here the full Iso-Seq ecosystem that enables researchers to achieve high-quality genome annotations. 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. Sequencing on a single SMRT Cell 8M on the Sequel II System produces up to 4 million full-length reads, sufficient to exhaustively characterize a whole transcriptome on the order of 15,000-17,000 genes with 100,000 or more transcripts. Most importantly, the method is supported by a maturing suite of official and community-developed tools. The SMRT Link Iso-Seq application outputs high-quality (>99% accurate), full-length transcript sequences that can optionally be mapped to a reference genome for a single SMRT Cell worth of data in 6-9 hours. For example, the SQANTI2 tool classifies Iso-Seq transcripts against a reference annotation, filters potential library artifacts, and processes information from both long read-only and short read-based quantification. IsoPhase is a tool for identifying allele-specific isoform expression. Cogent has been used to process Iso-Seq transcripts in a genome-independent manner to assess genome assemblies. Finally, IsoAnnot is an up-and-coming tool for identifying differential isoform expression across different samples. We describe how these tools complement each other and provide guidelines to make the best use out of Iso-Seq data for understanding transcriptomes.


February 5, 2021  |  

Movie: The new biology

This documentary film features the wave of cutting-edge technologies that now provide the opportunity to create predictive models of living systems, and gain wisdom about the fundamental nature of life…


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