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Auto Tag: Single-molecule real-time

June 1, 2021

High-quality de novo genome assembly and intra-individual mitochondrial instability in the critically endangered kakapo

The kakapo (Strigops habroptila) is a large, flightless parrot endemic to New Zealand. It is highly endangered with only ~150 individuals remaining, and intensive conservation efforts are underway to save this iconic species from extinction. These include genetic studies to understand critical genes relevant to fertility, adaptation and disease resistance, and genetic diversity across the remaining population for future breeding program decisions. To aid with these efforts, we have generated a high-quality de novo genome assembly using PacBio long-read sequencing. Using the new diploid-aware FALCON-Unzip assembler, the resulting genome of 1.06 Gb has a contig N50 of 5.6 Mb (largest contig 29.3 Mb), >350-times more contiguous compared to a recent short-read assembly of a closely related parrot (kea) species. We highlight the benefits of the higher contiguity and greater completeness of the kakapo genome assembly through examples of fully resolved genes important in wildlife conservation (contrasted with fragmented and incomplete gene resolution in short-read assemblies), in some cases even providing sequence for regions orthologous to gaps of missing sequence in the chicken reference genome. We also highlight the complete resolution of the kakapo mitochondrial genome, fully containing the mitochondrial control region which is missing from the previous dedicated kakapomitochondrial genome NCBI entry. For this region, we observed a marked heterogeneity in the number of tandem repeats in different mtDNAmolecules from a single bird tissue, highlighting the enhanced molecular resolution uniquely afforded by long-read, single-molecule PacBio sequencing.

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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).

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June 1, 2021

Amplification-free targeted enrichment and SMRT Sequencing of repeat-expansion genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease.

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June 1, 2021

SMRT-Cappable-seq reveals the complex operome of bacteria

SMRT-Cappable-seq combines the isolation of full-length prokaryotic primary transcripts with long read sequencing technology. It is the first experimental methodology to sequence entire prokaryotic transcripts. It identifies the transcription start site and termination site, thereby directly defines the operon structures genome-wide in prokaryotes. Applied to E.coli, SMRT-Cappable-seq identifies a total of ~2300 operons, among which ~900 are novel. Importantly, our result reveals a pervasive read-through of previous experimentally validated transcription termination sites. Termination read-through represents a powerful strategy to control gene expression. Taken together this data provides a first glance at the complexity of the ‘operome’ in bacteria and presents an invaluable resource for understanding gene regulation and function in bacteria.

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June 1, 2021

Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts

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 to the Iso-Seq bioinformatics pipeline increases the speed and scalability of data analysis while boosting the reliability of isoform detection and cross-platform usability. Here we report evaluation of Sequel Iso-Seq runs of human UHRR samples with spiked-in synthetic RNA controls and show that the new pipeline is more CPU efficient and recovers more human and synthetic isoforms while reducing the number of false positives. We also share the results of sequencing the well-characterized HCC-1954 breast cancer and normal breast cell lines, which will be made publicly available. Combined with the recent simplification of the Iso-Seq sample preparation2, the new analysis pipeline completes a streamlined workflow for revealing the most comprehensive picture of transcriptomes at the throughput needed to characterize cancer samples.

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June 1, 2021

Full-length cDNA sequencing of prokaryotic transcriptome and metatranscriptome samples

Next-generation sequencing has become a useful tool for studying transcriptomes. However, these methods typically rely on sequencing short fragments of cDNA, then attempting to assemble the pieces into full-length transcripts. Here, we describe a method that uses PacBio long reads to sequence full-length cDNAs from individual transcriptomes and metatranscriptome samples. We have adapted the PacBio Iso-Seq protocol for use with prokaryotic samples by incorporating RNA polyadenylation and rRNA-depletion steps. In conjunction with SMRT Sequencing, which has average readlengths of 10-15 kb, we are able to sequence entire transcripts, including polycistronic RNAs, in a single read. Here, we show full-length bacterial transcriptomes with the ability to visualize transcription of operons. In the area of metatranscriptomics, long reads reveal unambiguous gene sequences without the need for post-sequencing transcript assembly. We also show full-length bacterial transcripts sequenced after being treated with NEB’s Cappable-Seq, which is an alternative method for depleting rRNA and enriching for full-length transcripts with intact 5’ ends. Combining Cappable-Seq with PacBio long reads allows for the detection of transcription start sites, with the additional benefit of sequencing entire transcripts.

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June 1, 2021

Allelic specificity of immunoglobulin heavy chain (IGH@) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing

Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).

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June 1, 2021

The role of androgen receptor variant AR-V9 in prostate cancer

The expression of androgen receptor (AR) variants is a frequent, yet poorly-understood mechanism of clinical resistance to AR-targeted therapy for castration-resistant prostate cancer (CRPC). Among the multiple AR variants expressed in CRPC, AR-V7 is considered the most clinically-relevant AR variant due to broad expression in CRPC, correlations of AR-V7 expression with clinical resistance, and growth inhibition when AR-V7 is knocked down in CRPC models. Therefore, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The aim of this study was to understand whether other AR variants are co-expressed with AR-V7 and promote resistance to AR-targeted therapies. To test this, we utilized RNA-seq to characterize AR expression in CRPC models. RNA-seq revealed the frequent coexpression of AR-V9 and AR-V7 in multiple CRPC models and metastases. Furthermore, long-read single-molecule real-time (SMRT) sequencing of AR isoforms revealed that AR-V7 and AR-V9 shared a common 3’terminal cryptic exon. To test this, we knocked down AR-V7 in prostate cancer cell lines and confirmed that AR-V9 mRNA and protein expression were also impacted. In reporter assays with AR-responsive promoters, AR-V9 functioned as a constitutive activator of androgen/AR signaling. Similarly, infection of AR-V9 lentiviral construct in LNCaP cells induced androgen-independent cell proliferation. In conclusion, these data implicate co-expression of AR-V9 with AR-V7 as an important component of constitutive AR signaling and therapeutic resistance in CRPC.

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June 1, 2021

Best practices for whole genome sequencing using the Sequel System

Plant and animal whole genome sequencing has proven to be challenging, particularly due to genome size, high density of repetitive elements and heterozygosity. The Sequel System delivers long reads, high consensus accuracy and uniform coverage, enabling more complete, accurate, and contiguous assemblies of these large complex genomes. The latest Sequel chemistry increases yield up to 8 Gb per SMRT Cell for long insert libraries >20 kb and up to 10 Gb per SMRT Cell for libraries >40 kb. In addition, the recently released SMRTbell Express Template Prep Kit reduces the time (~3 hours) and DNA input (~3 µg), making the workflow easy to use for multi- SMRT Cell projects. Here, we recommend the best practices for whole genome sequencing and de novo assembly of complex plant and animal genomes. Guidelines for constructing large-insert SMRTbell libraries (>30 kb) to generate optimal read lengths and yields using the latest Sequel chemistry are presented. We also describe ways to maximize library yield per preparation from as littles as 3 µg of sheared genomic DNA. The combination of these advances makes plant and animal whole genome sequencing a practical application of the Sequel System.

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June 1, 2021

High-throughput SMRT Sequencing of clinically relevant targets

Targeted sequencing with Sanger as well as short read based high throughput sequencing methods is standard practice in clinical genetic testing. However, many applications beyond SNP detection have remained somewhat obstructed due to technological challenges. With the advent of long reads and high consensus accuracy, SMRT Sequencing overcomes many of the technical hurdles faced by Sanger and NGS approaches, opening a broad range of untapped clinical sequencing opportunities. Flexible multiplexing options, highly adaptable sample preparation method and newly improved two well-developed analysis methods that generate highly-accurate sequencing results, make SMRT Sequencing an adept method for clinical grade targeted sequencing. The Circular Consensus Sequencing (CCS) analysis pipeline produces QV 30 data from each single intra-molecular multi-pass polymerase read, making it a reliable solution for detecting minor variant alleles with frequencies as low as 1 %. Long Amplicon Analysis (LAA) makes use of insert spanning full-length subreads originating from multiple individual copies of the target to generate highly accurate and phased consensus sequences (>QV50), offering a unique advantage for imputation free allele segregation and haplotype phasing. Here we present workflows and results for a range of SMRT Sequencing clinical applications. Specifically, we illustrate how the flexible multiplexing options, simple sample preparation methods and new developments in data analysis tools offered by PacBio in support of Sequel System 5.1 can come together in a variety of experimental designs to enable applications as diverse as high throughput HLA typing, mitochondrial DNA sequencing and viral vector integrity profiling of recombinant adeno-associated viral genomes (rAAV).

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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.

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June 1, 2021

Single chromosomal genome assemblies on the Sequel System with Circulomics high molecular weight DNA extraction for microbes

Background: The Nanobind technology from Circulomics provides an elegant HMW DNA extraction solution for genome sequencing of Gram-positive and -negative microbes. Nanobind is a nanostructured magnetic disk that can be used for rapid extraction of high molecular weight (HMW) DNA from diverse sample types including cultured cells, blood, plant nuclei, and bacteria. Processing can be completed in <1 hour for most sample types and can be performed manually or automated with common instruments. Methods:We have validated several critical steps for generating high-quality microbial genome assemblies in a streamlined microbial multiplexing workflow. This new workflow enables high-volume, cost-effective sequencing of up to 16 microbes totaling 30 Mb in genome size on a single SMRT Cell 1M using a target shear size of 10 kb. We also evaluated this method on a pool of four “class 3” microbes that contain >7 kb repeats. Fragment size was increased to ~14 kb, with some fragments >30 kb. Results: Here we present a demonstration of these capabilities using isolates relevant to high-throughput sequencing applications, including common foodborne pathogens (Shigella, Listeria, Salmonella), and species often seen in hospital settings (Klebsiella, Staphylococcus). For nearly all microbes, including difficult-to-assemble class III microbes, we achieved complete de novo microbial assemblies of =5 chromosomal contigs with minimum quality scores of 40 (99.99% accuracy) using data from multiplexed SMRTbell libraries. Each library was sequenced on a single SMRT Cell 1M with the PacBio Sequel System and analyzed with streamlined SMRT Analysis assembly methods. Conclusions: We achieved high-quality, closed microbial genomes using a combination of Circulomics Nanobind extraction and PacBio SMRT Sequencing, along with a newly streamlined workflow that includes automated demultiplexing and push-button assembly.

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June 1, 2021

A simple segue from Sanger to high-throughput SMRT Sequencing with a M13 barcoding system

High-throughput NGS methods are increasingly utilized in the clinical genomics market. However, short-read sequencing data continues to remain challenged by mapping inaccuracies in low complexity regions or regions of high homology and may not provide adequate coverage within GC-rich regions of the genome. Thus, the use of Sanger sequencing remains popular in many clinical sequencing labs as the gold standard approach for orthogonal validation of variants and to interrogate missed regions poorly covered by second-generation sequencing. The use of Sanger sequencing can be less than ideal, as it can be costly for high volume assays and projects. Additionally, Sanger sequencing generates read lengths shorter than the region of interest, which limits its ability to accurately phase allelic variants. High-throughput SMRT Sequencing overcomes the challenges of both the first- and second-generation sequencing methods. PacBio’s long read capability allows sequencing of full-length amplicons

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June 1, 2021

FALCON-Phase integrates PacBio and HiC data for de novo assembly, scaffolding and phasing of a diploid Puerto Rican genome (HG00733)

Haplotype-resolved genomes are important for understanding how combinations of variants impact phenotypes. The study of disease, quantitative traits, forensics, and organ donor matching are aided by phased genomes. Phase is commonly resolved using familial data, population-based imputation, or by isolating and sequencing single haplotypes using fosmids, BACs, or haploid tissues. Because these methods can be prohibitively expensive, or samples may not be available, alternative approaches are required. de novo genome assembly with PacBio Single Molecule, Real-Time (SMRT) data produces highly contiguous, accurate assemblies. For non-inbred samples, including humans, the separate resolution of haplotypes results in higher base accuracy and more contiguous assembled sequences. Two primary methods exist for phased diploid genome assembly. The first, TrioCanu requires Illumina data from parents and PacBio data from the offspring. The long reads from the child are partitioned into maternal and paternal bins using parent-specific sequences; the separate PacBio read bins are then assembled, generating two fully phased genomes. An alternative approach (FALCON-Unzip) does not require parental information and separates PacBio reads, during genome assembly, using heterozygous SNPs. The length of haplotype phase blocks in FALCON-Unzip is limited by the magnitude and distribution of heterozygosity, the length of sequence reads, and read coverage. Because of this, FALCON-Unzip contigs typically contain haplotype-switch errors between phase blocks, resulting in primary contig of mixed parental origin. We developed FALCON-Phase, which integrates Hi-C data downstream of FALCON-Unzip to resolve phase switches along contigs. We applied the method to a human (Puerto Rican, HG00733) and non-human genome assemblies and evaluated accuracy using samples with trio data. In a cattle genome, we observe >96% accuracy in phasing when compared to TrioCanu assemblies as well as parental SNPs. For a high-quality PacBio assembly (>90-fold Sequel coverage) of a Puerto Rican individual we scaffolded the FALCON-Phase contigs, and re-phased the contigs creating a de novo scaffolded, phased diploid assembly with chromosome-scale contiguity.

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