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June 1, 2021  |  

Characterization of the Poly-T variants in the TOMM40 gene using PacBio long reads

Genes associated with several neurological disorders have been shown to be highly polymorphic. Targeted sequencing of these genes using NGS technologies is a powerful way to increase the cost-effectiveness of variant discovery and detection. However, for a comprehensive view of these target genes, it is necessary to have complete and uniform coverage across regions of interest. Unfortunately, short-read sequencing technologies are not ideal for these types of studies as they are prone to mis-mapping and often fail to span repetitive regions. Targeted sequencing with PacBio long reads provides the unique advantage of single-molecule observations of complex genomic regions. PacBio long reads not only provide continuous sequence data though polymorphic or repetitive regions, but also have no GC bias. Here we describe the characterization of the poly-T locus in TOMM40, a gene known to be associated with progression to Alzheimer’s, using PacBio long reads. Probes were designed to capture a 20 kb region comprising the TOMM40 and ApoE genes. Target regions were captured in multiple cell lines and sequencing libraries made using standard sample preparation methods. We will present our results on the poly-T structural variants that we observed in TOMM40 in these cell lines. We will also present our results on probe design optimization and barcoding strategies for a cost-effective solution.


June 1, 2021  |  

Phased human genome assemblies with Single Molecule, Real-Time Sequencing

In recent years, human genomic research has focused on comparing short-read data sets to a single human reference genome. However, it is becoming increasingly clear that significant structural variations present in individual human genomes are missed or ignored by this approach. Additionally, remapping short-read data limits the phasing of variation among individual chromosomes. This reduces the newly sequenced genome to a table of single nucleotide polymorphisms (SNPs) with little to no information as to the co-linearity (phasing) of these variants, resulting in a “mosaic” reference representing neither of the parental chromosomes. The variation between the homologous chromosomes is lost in this representation, including allelic variations, structural variations, or even genes present in only one chromosome, leading to lost information regarding allelic-specific gene expression and function. To address these limitations, we have made significant progress integrating haplotype information directly into genome assembly process with long reads. The FALCON-Unzip algorithm leverages a string graph assembly approach to facilitate identification and separation of heterozygosity during the assembly process to produce a highly contiguous assembly with phased haplotypes representing the genome in its diploid state. The outputs of the assembler are pairs of sequences (haplotigs) containing the allelic differences, including SNPs and structural variations, present in the two sets of chromosomes. The development and testing of our de-novo diploid assembler was facilitated and carefully validated using inbred reference model organisms and F1 progeny, which allowed us to ascertain the accuracy and concordance of haplotigs relative to the two inbred parental assemblies. Examination of the results confirmed that our haplotype-resolved assemblies are “Gold Level” reference genomes having a quality similar to that of Sanger-sequencing, BAC-based assembly approaches. We further sequenced and assembled two well-characterized human samples into their respective phased diploid genomes with gap-free contig N50 sizes greater than 23 Mb and haplotig N50 sizes greater than 380 kb. Results of these assemblies and a comparison between the haplotype sets are presented.


June 1, 2021  |  

Target enrichment using a neurology panel for 12 barcoded genomic DNA samples on the PacBio SMRT Sequencing platform

Target enrichment is a powerful tool for studies involved in understanding polymorphic SNPs with phasing, tandem repeats, and structural variations. With increasing availability of reference genomes, researchers can easily design a cost-effective targeted investigation with custom probes specific to regions of interest. Using PacBio long-read technology in conjunction with probe capture, we were able to sequence multi-kilobase enriched regions to fully investigate intronic and exonic regions, distinguish haplotypes, and characterize structural variations. Furthermore, we demonstrate this approach is advantageous for studying complex genomic regions previously inaccessible through other sequencing platforms. In the present work, 12 barcoded genomic DNA (gDNA) samples were sheared to 6 kb for target enrichment analysis using the Neurology panel provided by Roche NimbleGen. Probe-captured DNA was used to make SMRTbell libraries for SMRT Sequencing on the PacBio RS II. Our results demonstrate the ability to multiplex 12 samples and achieve 1300x enrichment of targeted regions. In addition, we achieved an even representation of on-target rate of 70% across the 12 barcoded genomic DNA samples.


June 1, 2021  |  

“SMRTer Confirmation”: Scalable clinical read-through variant confirmation using the Pacific Biosciences SMRT Sequencing platform

Next-generation sequencing (NGS) has significantly improved the cost and turnaround time for diagnostic genetic tests. ACMG recommends variant confirmation by an orthogonal method, unless sufficiently high sensitivity and specificity can be demonstrated using NGS alone. Most NGS laboratories make extensive use of Sanger sequencing for secondary confirmation of single nucleotide variants (SNVs) and indels, representing a large fraction of the cost and time required to deliver high quality genetic testing data to clinicians and patients. Despite its established data quality, Sanger is not a high-throughput method by today’s standards from either an assay or analysis standpoint as it can involve manual review of Sanger traces and is not amenable to multiplexing. Toward a scalable solution for confirmation, Invitae has developed a fully automated and LIMS-tracked assay and informatics pipeline that utilizes the Pacific Biosciences SMRT sequencing platform. Invitae’s pipeline generates PCR amplicons that encompass the variant(s) of interest, which are converted to closed DNA structures (SMRTbells) and sequenced in pools of 96 per SMRTcell. Each amplicon is appended with a 16nt barcode that encodes the patient and variant IDs. Per-sample de-multiplexing, alignment, variant calling, and confirmation resolution are handled via an automated pipeline. The confirmation process was validated by analyzing 243 clinical SNVs and indels in parallel with the gold standard Sanger sequencing method. Amplicons were sequenced and analyzed in technical replicates to demonstrate reproducibility. In this study, the PacBio-based confirmation pipeline demonstrated high reproducibility (97.5%), and outperformed Sanger in the fraction of primary NGS variants confirmed (PacBio = 93.4% and 94.7% confirmed across two replicates, Sanger = 84.8%) while having 100% concordance of confirmation status among overlapping confirmation calls.


June 1, 2021  |  

Profiling complex population genomes 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. These results illustrate ways in which long accurate reads benefit analysis of complex communities.


June 1, 2021  |  

High-quality, highly contiguous re-assembly of the pig genome

Many applications of high throughput sequencing rely on the availability of an accurate reference genome. Errors in the reference genome assembly increase the number of false-positives in downstream analyses. Recently, we have shown that over 33% of the current pig reference genome, Sscrofa10.2, is either misassembled or otherwise unreliable for genomic analyses. Additionally, ~10% of the bases in the assembly are Ns in gaps of an arbitrary size. Thousands of highly fragmented contigs remain unplaced and many genes are known to be missing from the assembly. Here we present a new assembly of the pig genome, Sscrofa11, assembled using 65X PacBio sequencing from T.J. Tabasco, the same Duroc sow used in the assembly of Sscrofa10.2. The PacBio reads were assembled using the Falcon assembly pipeline resulting in 3,206 contigs with an initial contig N50 of 14.5Mb. We used Sscrofa10.2 as a template to scaffold the PacBio contigs, under the assumption that its gross structure is correct, and used PBJelly to fill gaps. Additional gaps were filled using large, sequenced BACs from the original assembly. Following gap filling, the assembly has substantially improved contiguity and contains more sequence than the Sscrofa10.2 assembly. Arrow and Pilon were used to polish the assembly. The contig N50 is now 58.5Mb with 103 gaps remaining. By comparing regions of the two assemblies we show that regions with structural abnormalities we identified in Sscrofa10.2 are resolved in the new PacBio assembly.


June 1, 2021  |  

Screening and characterization of causative structural variants for bipolar disorder in a significantly linked chromosomal region onXq24-q27 in an extended pedigree from a genetic isolate

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.


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.


June 1, 2021  |  

De novo PacBio long-read assembled avian genomes correct and add to genes important in neuroscience and conservation research

To test the impact of high-quality genome assemblies on biological research, we applied PacBio long-read sequencing in conjunction with the new, diploid-aware FALCON-Unzip assembler to a number of bird species. These included: the zebra finch, for which a consortium-generated, Sanger-based reference exists, to determine how the FALCON-Unzip assembly would compare to the current best references available; Anna’s hummingbird genome, which had been assembled with short-read sequencing methods as part of the Avian Phylogenomics phase I initiative; and two critically endangered bird species (kakapo and ‘alala) of high importance for conservations efforts, whose genomes had not previously been sequenced and assembled.


June 1, 2021  |  

A high-quality genome assembly of SMRT sequences reveals long range haplotype structure in the diploid mosquito Aedes aegypti

Aedes aegypti is a tropical and subtropical mosquito vector for Zika, yellow fever, dengue fever, and chikungunya. We describe the first diploid assembly of an insect genome, using SMRT Sequencing and the open-source assembler FALCON-Unzip. This assembly has high contiguity (contig N50 1.3 Mb), is more complete than previous assemblies (Length 1.45 Gb with 87% BUSCO genes complete), and is high quality (mean base >QV30 after polishing). Long-range haplotype structure, in some cases encompassing more than 4 Mb of extremely divergent homologous sequence with dramatic differences in coding sequence content, is resolved using a combination of the FALCON-Unzip assembler, genome annotation, coverage depth, and pairwise nucleotide alignments.


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.


June 1, 2021  |  

A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes such as APP, PSEN1 and PSEN2 that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form of late-onset AD. However, the identified SNPs are typically not the actual causal variants, but are in linkage disequilibrium with the presumed causative variant (Van Cauwenberghe C, et al., The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med 2015;18:421-430).


June 1, 2021  |  

Structural variant detection with low-coverage Pacbio sequencing

Despite amazing progress over the past quarter century in the technology to detect genetic variants, intermediate-sized structural variants (50 bp to 50 kb) have remained difficult to identify. Such variants are too small to detect with array comparative genomic hybridization, but too large to reliably discover with short-read DNA sequencing. Recent de novo assemblies of human genomes have demonstrated the power of PacBio Single Molecule, Real-Time (SMRT) Sequencing to fill this technology gap and sensitively identify structural variants in the human genome. While de novo assembly is the ideal method to identify variants in a genome, it requires high depth of coverage. A structural variant discovery approach that utilizes lower coverage would facilitate evaluation of large patient and population cohorts. Here we introduce such an approach and apply it to 10-fold coverage of several human genomes generated on the PacBio Sequel System. To identify structural variants in low-fold coverage whole genome sequencing data, we apply a reference-based, re-sequencing workflow. First, reads are mapped to the human reference genome with a local aligner. The local alignments often end at structural variant loci. To connect co-linear local alignments across structural variants, we apply a novel algorithm that merges alignments into “chains” and refines the alignment edges. Then, the chained alignments are scanned for windows with an excess of insertions or deletions to identify candidate structural variant loci. Finally, the read support at each putative variant locus is evaluated to produce a variant call. Single nucleotide information is incorporated to phase and evaluate the zygosity of each structural variant. In 10-fold coverage human genome sequence, we identify the vast majority of the structural variants found by de novo assembly, thus demonstrating the power of low-fold coverage SMRT Sequencing to affordably and effectively detect structural variants.


June 1, 2021  |  

Screening for causative structural variants in neurological disorders using long-read sequencing

Over the past decades neurological disorders have been extensively studied producing a large number of candidate genomic regions and candidate genes. The SNPs identified in these studies rarely represent the true disease-related functional variants. However, more recently a shift in focus from SNPs to larger structural variants has yielded breakthroughs in our understanding of neurological disorders.Here we have developed candidate gene screening methods that combine enrichment of long DNA fragments with long-read sequencing that is optimized for structural variation discovery. We have also developed a novel, amplification-free enrichment technique using the CRISPR/Cas9 system to target genomic regions.We sequenced gDNA and full-length cDNA extracted from the temporal lobe for two Alzheimer’s patients for 35 GWAS candidate genes. The multi-kilobase long reads allowed for phasing across the genes and detection of a broad range of genomic variants including SNPs to multi-kilobase insertions, deletions and inversions. In the full-length cDNA data we detected differential allelic isoform complexity, novel exons as well as transcript isoforms. By combining the gDNA data with full-length isoform characterization allows to build a more comprehensive view of the underlying biological disease mechanisms in Alzheimer’s disease. Using the novel PCR-free CRISPR-Cas9 enrichment method we screened several genes including the hexanucleotide repeat expansion C9ORF72 that is associated with 40% of familiar ALS cases. This method excludes any PCR bias or errors from an otherwise hard to amplify region as well as preserves the basemodication in a single molecule fashion which allows you to capture mosaicism present in the sample.


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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