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June 1, 2021  |  

Characterizing haplotype diversity at the immunoglobulin heavy chain locus across human populations using novel long-read sequencing and assembly approaches

The human immunoglobulin heavy chain locus (IGH) remains among the most understudied regions of the human genome. Recent efforts have shown that haplotype diversity within IGH is elevated and exhibits population specific patterns; for example, our re-sequencing of the locus from only a single chromosome uncovered >100 Kb of novel sequence, including descriptions of six novel alleles, and four previously unmapped genes. Historically, this complex locus architecture has hindered the characterization of IGH germline single nucleotide, copy number, and structural variants (SNVs; CNVs; SVs), and as a result, there remains little known about the role of IGH polymorphisms in inter-individual antibody repertoire variability and disease. To remedy this, we are taking a multi-faceted approach to improving existing genomic resources in the human IGH region. First, from whole-genome and fosmid-based datasets, we are building the largest and most ethnically diverse set of IGH reference assemblies to date, by employing PacBio long-read sequencing combined with novel algorithms for phased haplotype assembly. In total, our effort will result in the characterization of >15 phased haplotypes from individuals of Asian, African, and European descent, to be used as a representative reference set by the genomics and immunogenetics community. Second, we are utilizing this more comprehensive sequence catalogue to inform the design and analysis of novel targeted IGH genotyping assays. Standard targeted DNA enrichment methods (e.g., exome capture) are currently optimized for the capture of only very short (100’s of bp) DNA segments. Our platform uses a modified bench protocol to pair existing capture-array technologies with the enrichment of longer fragments of DNA, enabling the use of PacBio sequencing of DNA segments up to 7 Kb. This substantial increase in contiguity disambiguates many of the complex repeated structures inherent to the locus, while yielding the base pair fidelity required to call SNVs. Together these resources will establish a stronger framework for further characterizing IGH genetic diversity and facilitate IGH genomic profiling in the clinical and research settings, which will be key to fully understanding the role of IGH germline variation in antibody repertoire development and disease.

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June 1, 2021  |  

Structural variant combining Illumina and low-coverage PacBio

Structural variant calling combining Illumina and low-coverage Pacbio Detection of large genomic variation (structural variants) has proven challenging using short-read methods. Long-read approaches which can span these large events have promise to dramatically expand the ability to accurately call structural variants. Although sequencing with Pacific Biosciences (Pacbio) long-read technology has become increasingly high throughput, generating high coverage with the technology can still be limiting and investigators often would like to know what pacbio coverages are adequate to call structural variants. Here, we present a method to identify a substantially higher fraction of structural variants in the human genome using low-coverage pacbio data by multiple strategies for ensembling data types and algorithms. Algorithmically, we combine three structural variant callers: PBHoney by Adam English, Sniffles by Fritz Sedlazeck, and Parliament by Adam English (which we have modified to improve for speed). Parliament itself uses a combination of Pacbio and Illumina data with a number of short-read callers (Breakdancer, Pindel, Crest, CNVnator, Delly, and Lumpy). We show that the outputs of these three programs are largely complementary to each other, with each able to uniquely access different sets of structural variants at different coverages. Combining them together can more than double the recall of true structural variants from a truth set relative to sequencing with Illumina alone, with substantial improvements even at low pacbio coverages (3x – 7x). This allows us to present for the first time cost-benefit tradeoffs to investigators about how much pacbio sequencing will yield what improvements in SV-calling. This work also builds upon the foundational work of Genome in a Bottle led by Justin Zook in establishing a truth set for structural variants in the Ashkenazim-Jewish trio data recently released. This work demonstrates the power of this benchmark set – one of the first of its kind for structural variation data – to help understand and refine the accuracies of calling structural variants with a number of approaches.

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June 1, 2021  |  

Effect of coverage depth and haplotype phasing on structural variant detection with PacBio long reads

Each human genome has thousands of structural variants compared to the reference assembly, up to 85% of which are difficult or impossible to detect with Illumina short reads and are only visible with long, multi-kilobase reads. The PacBio RS II and Sequel single molecule, real-time (SMRT) sequencing platforms have made it practical to generate long reads at high throughput. These platforms enable the discovery of structural variants just as short-read platforms did for single nucleotide variants. Numerous software algorithms call structural variants effectively from PacBio long reads, but algorithm sensitivity is lower for insertion variants and all heterozygous variants. Furthermore, the impact of coverage depth and read lengths on sensitivity is not fully characterized. To quantify how zygosity, coverage depth, and read lengths impact the sensitivity of structural variant detection, we obtained high coverage PacBio sequences for three human samples: haploid CHM1, diploid NA12878, and diploid SK-BR-3. For each dataset, reads were randomly subsampled to titrate coverage from 0.5- to 50-fold. The structural variants detected at each coverage were compared to the set at “full” 50-fold coverage. For the diploid samples, additional titrations were performed with reads first partitioned by phase using single nucleotide variants for essentially haploid structural variant discovery. Even at low coverages (1- to 5-fold), PacBio long reads reveal hundreds of structural variants that are not seen in deep 50-fold Illumina whole genome sequences. At moderate 10-fold PacBio coverage, a majority of structural variants are detected. Sensitivity begins to level off at around 40-fold coverage, though it does not fully saturate before 50-fold. Phasing improves sensitivity for all variant types, especially at moderate 10- to 20-fold coverage. Long reads are an effective tool to identify and phase structural variants in the human genome. The majority of variants are detected at moderate 10-fold coverage, and even extremely low long-read coverage (1- to 5-fold) reveals variants that are invisible to short-read sequencing. Performance will continue to improve with better software and longer reads, which will empower studies to connect structural variants to healthy and disease traits in the human population.

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June 1, 2021  |  

The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations

The major histocompatibility complex (MHC), or human leukocyte antigen (HLA) in humans, is a highly diverse gene family with a key role in immune response to disease; and has been implicated in auto-immune disease, cancer, infectious disease susceptibility, and vaccine response. It has clinical importance in the field of solid organ and bone marrow transplantation, where donors and recipient matching of HLA types is key to transplanted organ outcomes. The Sanger based typing (SBT) methods currently used in clinical practice do not capture the full diversity across this region, and require specific reference sequences to deconvolute ambiguity in HLA types. However, reference databases are based largely on European populations, and the full extent of diversity in Africa remains poorly understood. Here, we present the first systematic characterisation of HLA diversity within Africa in the pilot phase of the MHC Diversity in Africa Project, together with an evaluation of methods to carry out scalable cost-effective, as well as reliable, typing of this region in African populations.To sample a geographically representative panel of African populations we obtained 125 samples, 25 each from the Zulu (South Africa), Igbo (Nigeria), Kalenjin (Kenya), Moroccan and Ashanti (Ghana) groups. For methods validation we included two controls from the International Histocompatibility Working Group (IHWG) collection with known typing information. Sanger typing and Illumina HiSeq X sequencing of these samples indicated potentially novel Class I and Class II alleles; however, we found poor correlation between HiSeq X sequencing and SBT for both classes. Long Range PCR and high resolution PacBio RS-II typing of 4 of these samples identified 7 novel Class II alleles, highlighting the high levels of diversity in these populations, and the need for long read sequencing approaches to characterise this comprehensively. We have now expanded this approach to the entire pilot set of 125 samples. We present these confirmed types and discuss a workflow for scaling this to 5000 individuals across Africa.The large number of new alleles identified in our pilot suggests the high level of African HLA diversity and the utility of high resolution methods. The MDAP project will provide a framework for accurate HLA typing, in addition to providing an invaluable resource for imputation in GWAS, boosting power to identify and resolve HLA disease associations.

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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.

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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.

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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.

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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.

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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).

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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.

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June 1, 2021  |  

SMRT Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants

Prostate cancer is the most frequently diagnosed male cancer. For prostate cancer that has progressed to an advanced or metastatic stage, androgen deprivation therapy (ADT) is the standard of care. ADT inhibits activity of the androgen receptor (AR), a master regulator transcription factor in normal and cancerous prostate cells. The major limitation of ADT is the development of castration-resistant prostate cancer (CRPC), which is almost invariably due to transcriptional re-activation of the AR. One mechanism of AR transcriptional re-activation is expression of AR-V7, a truncated, constitutively active AR variant (AR-V) arising from alternative AR pre-mRNA splicing. Noteworthy, AR-V7 is being developed as a predictive biomarker of primary resistance to androgen receptor (AR)-targeted therapies in CRPC. Multiple additional AR-V species are expressed in clinical CRPC, but the extent to which these may be co-expressed with AR-V7 or predict resistance is not known.

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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.

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June 1, 2021  |  

Detecting pathogenic structural variants with low-coverage PacBio sequencing.

Though a role for structural variants in human disease has long been recognized, it has remained difficult to identify intermediate-sized variants (50 bp to 5 kb), which are too small to detect with array comparative genomic hybridization, but too large to reliably discover with short-read DNA sequencing. Recent studies have demonstrated that PacBio Single Molecule, Real-Time (SMRT) sequencing fills this technology gap. SMRT sequencing detects tens of thousands of structural variants in the human genome, approximately five times the sensitivity of short-read DNA sequencing.

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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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