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Asset Tag: SMRT Technology,

June 1, 2021  |  

Profiling complex communities with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS sequencing enables functional profiling as well, with the ultimate goal of complete genome assemblies. Here we compare the complex microbiomes in 5 cow rumen samples, for which Illumina WGS sequence data was also available. To maximize the PacBio single-molecule sequence accuracy, libraries of 2 to 3 kb were generated, allowing many polymerase passes per molecule. The resulting reads were filtered at predicted single-molecule accuracy levels up to 99.99%. Community compositions of the 5 samples were compared with Illumina WGS assemblies from the same set of samples, indicating rare organisms were often missed with Illumina. Assembly from PacBio CCS reads yielded a contig >100 kb in length with 6-fold coverage. Mapping of Illumina reads to the 101 kb contig verified the PacBio assembly and contig sequence. Scaffolding with reads from a PacBio unsheared library produced a complete genome of 2.4 Mb. These results illustrate ways in which long accurate reads benefit analysis of complex communities.

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June 1, 2021  |  

T-cell receptor profiling using PacBio sequencing of SMARTer libraries

T-cells play a central part in the immune response in humans and related species. T-cell receptors (TCRs), heterodimers located on the T-cell surface, specifically bind foreign antigens displayed on the MHC complex of antigen-presenting cells. The wide spectrum of potential antigens is addressed by the diversity of TCRs created by V(D)J recombination. Profiling this repertoire of TCRs could be useful from, but not limited to, diagnosis, monitoring response to treatments, and examining T-cell development and diversification.

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June 1, 2021  |  

Structural variant detection with low-coverage PacBio sequencing

Structural variants (genomic differences =50 base pairs) contribute to the evolution of organisms traits and human disease. Most structural variants (SVs) are too small to detect with array comparative genomic hybridization but too large to reliably discover with short-read DNA sequencing. Recent studies in human genomes show that PacBio SMRT Sequencing sensitively detects structural variants.

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June 1, 2021  |  

De novo assembly and preliminary annotation of the Schizocardium californicum genome

Animals in the phylum Hemichordata have provided key understanding of the origins and development of body patterning and nervous system organization. However, efforts to sequence and assemble the genomes of highly heterozygous non-model organisms have proven to be difficult with traditional short read approaches. Long repetitive DNA structures, extensive structural variation between haplotypes in polyploid species, and large genome sizes are limiting factors to achieving highly contiguous genome assemblies. Here we present the highly contiguous de novo assembly and preliminary annotation of an indirect developing hemichordate genome, Schizocardium californicum, using SMRT Sequening long reads.

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June 1, 2021  |  

Targeted sequencing using a long-read sequencing technology

Targeted sequencing employing PCR amplification is a fundamental approach to studying human genetic disease. PacBio’s Sequel System and supporting products provide an end-to-end solution for amplicon sequencing, offering better performance to Sanger technology in accuracy, read length, throughput, and breadth of informative data. Sample multiplexing is supported with three barcoding options providing the flexibility to incorporate unique sample identifiers during target amplification or library preparation. Multiplexing is key to realizing the full capacity of the 1 million individual reactions per Sequel SMRT Cell. Two analysis workflows that can generate high-accuracy results support a wide range of amplicon sizes in two ranges from 250 bp to 3 kb and from 3 kb to >10 kb. The Circular Consensus Sequencing workflow results in high accuracy through intra-molecular consensus generation, while high accuracy for the Long Amplicon Analysis workflow is achieved by clustering of individual long reads from multiple reactions. Here we present workflows and results for single- molecule sequencing of amplicons for human genetic analysis.

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June 1, 2021  |  

Haplotyping of full-length transcript reads from long-read sequencing can reveal allelic imbalances in isoform expression

The Pacific Biosciences Iso-Seq method, which can produce high-quality isoform sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. Here, we develop an algorithm called IsoPhase that postprocesses Iso-Seq data to retrieve allele specific isoform information. Using simulated data, we show that for both diploid and tetraploid genomes, IsoPhase results in good SNP recovery with low FDR at error rates consistent with CCS reads. We apply IsoPhase to a haplotyperesolved genome assembly and multiple fetal tissue Iso-Seq dataset from a F1 cross of Angus x Brahman cattle subspecies. IsoPhase-called haplotypes were validated by the phased assembly and demonstrate the potential for revealing allelic imbalances in isoform expression.

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June 1, 2021  |  

Mitochondrial DNA sequencing using PacBio SMRT technology

Mitochondrial DNA (mtDNA) is a compact, double-stranded circular genome of 16,569 bp with a cytosine-rich light (L) chain and a guanine-rich heavy (H) chain. mtDNA mutations have been increasingly recognized as important contributors to an array of human diseases such as Parkinson’s disease, Alzheimer’s disease, colorectal cancer and Kearns–Sayre syndrome. mtDNA mutations can affect all of the 1000-10,000 copies of the mitochondrial genome present in a cell (homoplasmic mutation) or only a subset of copies (heteroplasmic mutation). The ratio of normal to mutant mtDNAs within cells is a significant factor in whether mutations will result in disease, as well as the clinical presentation, penetrance, and severity of the phenotype. Over time, heteroplasmic mutations can become homoplastic due to differential replication and random assortment. Full characterization of the mitochondrial genome would involve detection of not only homoplastic but heteroplasmic mutations, as well as complete phasing. Previously, we sequenced human mtDNA on the PacBio RS II System with two partially overlapping amplicons. Here, we present amplification-free, full-length sequencing of linearized mtDNA using the Sequel System. Full-length sequencing allows variant phasing along the entire mitochondrial genome, identification of heteroplasmic variants, and detection of epigenetic modifications that are lost in amplicon-based methods.

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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  |  

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  |  

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  |  

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  |  

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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