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Asset Tag: haplotype

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  |  

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  |  

Phased diploid genome assembly with single-molecule real-time sequencing

While genome assembly projects have been successful in many haploid and inbred species, the assembly of non-inbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.

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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, chikungunya, and other diseases. The outbreak of Zika in the Americas, which can cause microcephaly in the fetus of infected women, adds urgency to the need for a high-quality reference genome in order to better understand the organism’s biology and its role in transmitting human disease. 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). Long-range haplotype structure, in some cases encompassing more than 4 Mb of extremely divergent homologous sequence, 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  |  

Detecting pathogenic structural variants with long-read PacBio SMRT Sequencing

Most of the base pairs that differ between two human genomes are in intermediate-sized structural variants (50 bp to 5 kb), which are too small to detect with array comparative genomic hybridization or optical mapping but too large to reliably discover with short-read DNA sequencing. Long-read sequencing with PacBio Single Molecule, Real-Time (SMRT) Sequencing platforms fills this technology gap. PacBio SMRT Sequencing detects tens of thousands of structural variants in a human genome with approximately five times the sensitivity of short-read DNA sequencing. Effective application of PacBio SMRT Sequencing to detect structural variants requires quality bioinformatics tools that account for the characteristics of PacBio reads. To provide such a solution, we developed pbsv, a structural variant caller for PacBio reads that works as a chain of simple stages: 1) map reads to the reference genome, 2) identify reads with signatures of structural variation, 3) cluster nearby reads with similar signatures, 4) summarize each cluster into a consensus variant, and 5) filter for variants with sufficient read support. To evaluate the baseline performance of pbsv, we generated high coverage of a diploid human genome on the PacBio Sequel System, established a target set of structural variants, and then titrated to lower coverage levels. The false discovery rate for pbsv is low at all coverage levels. Sensitivity is high even at modest coverage: above 85% at 10-fold coverage and above 95% at 20-fold coverage. To assess the potential for PacBio SMRT Sequencing to identify pathogenic variants, we evaluated an individual with clinical symptoms suggestive of Carney complex for whom short-read whole genome sequencing was uninformative. The individual was sequenced to 9-fold coverage on the PacBio Sequel System, and structural variants were called with pbsv. Filtering for rare, genic structural variants left six candidates, including a heterozygous 2,184 bp deletion that removes the first coding exon of PRKAR1A. Null mutations in PRKAR1Acause autosomal dominant Carney complex, type 1. The variant was determined to be de novo, and it was classified as likely pathogenic based on ACMG standards and guidelines for variant interpretation. These case studies demonstrate the ability of pbsv to detect structural variants in low-coverage PacBio SMRT Sequencing and suggest the importance of considering structural variants in any study of human genetic variation.

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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  |  

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  |  

Best practices for diploid assembly of complex genomes using PacBio: A case study of Cascade Hops

A high quality reference genome is an essential resource for plant and animal breeding and functional and evolutionary studies. The common hop (Humulus lupulus, Cannabaceae) is an economically important crop plant used to flavor and preserve beer. Its genome is large (flow cytometrybased estimates of diploid length >5.4Gb1), highly repetitive, and individual plants display high levels of heterozygosity, which make assembly of an accurate and contiguous reference genome challenging with conventional short-read methods. We present a contig assembly of Cascade Hops using PacBio long reads and the diploid genome assembler, FALCON-Unzip2. The assembly has dramatically improved contiguity and completeness over earlier short-read assemblies. The genome is primarily assembled as haplotypes due to the outbred nature of the organism. We explore patterns of haplotype divergence across the assembly and present strategies to deduplicate haplotypes prior to scaffolding

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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  |  

Haplotyping using full-length transcript sequencing reveals allele-specific expression

An important need in analyzing complex genomes is the ability to separate and phase haplotypes. While whole genome assembly can deliver this information, it cannot reveal whether there is allele-specific gene or isoform expression. The PacBio Iso-Seq method, which can produce high-quality transcript sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. We present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents and two F1 cross hybrids. We validated the majority of the SNPs called with IsoPhase against matching short read data and identified cases of allele-specific, gene-level and isoform-level expression.

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