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Auto Tag: Haplotyping

June 1, 2021  |  

Advances in sequence consensus and clustering algorithms for effective de novo assembly and haplotyping applications.

One of the major applications of DNA sequencing technology is to bring together information that is distant in sequence space so that understanding genome structure and function becomes easier on a large scale. The Single Molecule Real Time (SMRT) Sequencing platform provides direct sequencing data that can span several thousand bases to tens of thousands of bases in a high-throughput fashion. In contrast to solving genomic puzzles by patching together smaller piece of information, long sequence reads can decrease potential computation complexity by reducing combinatorial factors significantly. We demonstrate algorithmic approaches to construct accurate consensus when the differences between reads are dominated by insertions and deletions. High-performance implementations of such algorithms allow more efficient de novo assembly with a pre-assembly step that generates highly accurate, consensus-based reads which can be used as input for existing genome assemblers. In contrast to recent hybrid assembly approach, only a single ~10 kb or longer SMRTbell library is necessary for the hierarchical genome assembly process (HGAP). Meanwhile, with a sensitive read-clustering algorithm with the consensus algorithms, one is able to discern haplotypes that differ by less than 1% different from each other over a large region. One of the related applications is to generate accurate haplotype sequences for HLA loci. Long sequence reads that can cover the whole 3 kb to 4 kb diploid genomic regions will simplify the haplotyping process. These algorithms can also be applied to resolve individual populations within mixed pools of DNA molecules that are similar to each, e.g., by sequencing viral quasi-species samples.

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June 1, 2021  |  

Genomic Architecture of the KIR and MHC-B and -C Regions in Orangutan

PacBio 2013 User Group Meeting Presentation Slides: Lisbeth Guethlein from Stanford University School of Medicine looked at highly repetitive and variable immune regions of the orangutan genome. Guethlein reported that “PacBio managed to accomplish in a week what I have been working on for a couple years” (with Sanger sequencing), and the results were concordant. “Long story short, I was a happy customer.”

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June 1, 2021  |  

A novel analytical pipeline for de novo haplotype phasing and amplicon analysis using SMRT Sequencing technology.

While the identification of individual SNPs has been readily available for some time, the ability to accurately phase SNPs and structural variation across a haplotype has been a challenge. With individual reads of an average length of 9 kb (P5-C3), and individual reads beyond 30 kb in length, SMRT Sequencing technology allows the identification of mutation combinations such as microdeletions, insertions, and substitutions without any predetermined reference sequence. Long- amplicon analysis is a novel protocol that identifies and reports the abundance of differing clusters of sequencing reads within a single library. Graphs generated via hierarchical clustering of individual sequencing reads are used to generate Markov models representing the consensus sequence of individual clusters found to be significantly different. Long-amplicon analysis is capable of differentiating between underlying sequences that are 99.9% similar, which is suitable for haplotyping and differentiating pseudogenes from coding transcripts. This protocol allows for the identification of structural variation in the MUC5AC gene sequence, despite the presence of a gap in the current genome assembly, and can also be used for HLA haplotyping. Clustering can also been applied to identify full length transcripts for the purpose of estimating consensus sequences and enumerating isoform types. Long-amplicon analysis allows for the elucidation of complex regions otherwise missed by other sequencing technologies, which may contribute to the diagnosis and understanding of otherwise complex diseases.

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June 1, 2021  |  

Unique haplotype structure determination in human genome using Single Molecule, Real-Time (SMRT) Sequencing of targeted full-length fosmids.

Determination of unique individual haplotypes is an essential first step toward understanding how identical genotypes having different phases lead to different biological interpretations of function, phenotype, and disease. Genome-wide methods for identifying individual genetic variation have been limited in their ability to acquire phased, extended, and complete genomic sequences that are long enough to assemble haplotypes with high confidence. We explore a recombineering approach for isolation and sequencing of a tiling of targeted fosmids to capture interesting regions from human genome. Each individual fosmid contains large genomic fragments (~35?kb) that are sequenced with long-read SMRT technology to generate contiguous long reads. These long reads can be easily de novo assembled for targeted haplotype resolution within an individual’s genomes. The P5-C3 chemistry for SMRT Sequencing generated contiguous, full-length fosmid sequences of 30 to 40 kb in a single read, allowing assembly of resolved haplotypes with minimal data processing. The phase preserved in fosmid clones spanned at least two heterozygous variant loci, providing the essential detail of precise haplotype structures. We show complete assembly of haplotypes for various targeted loci, including the complex haplotypes of the KIR locus (~150 to 200 kb) and conserved extended haplotypes (CEHs) of the MHC region. This method is easily applicable to other regions of the human genome, as well as other genomes.

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June 1, 2021  |  

Targeted SMRT Sequencing and phasing using Roche NimbleGen’s SeqCap EZ enrichment

As a cost-effective alternative to whole genome human sequencing, targeted sequencing of specific regions, such as exomes or panels of relevant genes, has become increasingly common. These methods typically include direct PCR amplification of the genomic DNA of interest, or the capture of these targets via probe-based hybridization. Commonly, these approaches are designed to amplify or capture exonic regions and thereby result in amplicons or fragments that are a few hundred base pairs in length, a length that is well-addressed with short-read sequencing technologies. These approaches typically provide very good coverage and can identify SNPs in the targeted region, but are unable to haplotype these variants. Here we describe a targeted sequencing workflow that combines Roche NimbleGen’s SeqCap EZ enrichment technology with Pacific Biosciences’ SMRT Sequencing to provide a more comprehensive view of variants and haplotype information over multi-kilobase regions. While the SeqCap EZ technology is typically used to capture 200 bp fragments, we demonstrate that 6 kb fragments can also be utilized to enrich for long fragments that extend beyond the targeted capture site and well into (and often across) the flanking intronic regions. When combined with the long reads of SMRT Sequencing, multi-kilobase regions of the human genome can be phased and variants detected in exons, introns and intergenic regions.

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June 1, 2021  |  

Complete resequencing of extended genomic regions using fosmid target capture and single molecule real-time (SMRT) long read sequencing technology.

A longstanding goal of genomic analysis is the identification of causal genetic factors contributing to disease. While the common disease/common variant hypothesis has been tested in many genome-wide association studies, few advancements in identifying causal variation have been realized, and instead recent findings point away from common variants towards aggregate rare variants as causal. A challenge is obtaining complete phased genomic sequences over extended genomic regions from sufficient numbers of cases and controls to identify all potential variation causal of a disease. To address this, we modified methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generaton that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As proof of principal, we validated the approach by resequencing four 800 kbp segments that span a major histocompatibility complex (MHC) common extended haplotype (CEH) associated with disease. The data revealed the extent of conservation exposing a near identity among four DR4 CEHs over conserved regions, detailing rare variation and measuring sequence accuracy. In a second test, we sequenced the complete KIR haplotypes from 8 individuals within a specific timeframe and cost. Single molecule long-read sequencing technology generated contiguous full-­length fosmid sequences of 30 to 40 kb in a single read, allowing assembly of resolved haplotypes with very little data processing. All of the sequences produced from these projects were contiguous, phased, with accuracy above 99.99%. The results demonstrated that cost-effective scale-­up is possible to generate scores to hundreds of phased chromosomal sequences of extended lengths that can encompass genomic regions associated with disease.

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June 1, 2021  |  

Highly contiguous de novo human genome assembly and long-range haplotype phasing using SMRT Sequencing

The long reads, random error, and unbiased sampling of SMRT Sequencing enables high quality, de novo assembly of the human genome. PacBio long reads are capable of resolving genomic variations at all size scales, including SNPs, insertions, deletions, inversions, translocations, and repeat expansions, all of which are important in understanding the genetic basis for human disease and difficult to access via other technologies. In demonstration of this, we report a new high-quality, diploid aware de novo assembly of Craig Venter’s well-studied genome.

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June 1, 2021  |  

Assembly of complete KIR haplotypes from a diploid individual by the direct sequencing of full-length fosmids.

We show that linearizing and directly sequencing full-length fosmids simplifies the assembly problem such that it is possible to unambiguously assemble individual haplotypes for the highly repetitive 100-200 kb killer Ig-like receptor (KIR) gene loci of chromosome 19. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the KIR locus, means that sequence assembly of pooled samples into complete haplotypes is difficult and in many cases impossible. The current maximum read length generated by SMRT Sequencing exceeds the length of a 40 kb fosmid; it is therefore possible to span an entire fosmid in one sequencing read. Shearing, sequencing and assembling fosmids in a shotgun approach is prone to errors when the underlying sequence is highly repetitive. We show that it is possible to directly sequence linearized fosmids and generate a high-quality consensus by simple alignment, removing the need for an error-prone assembly step. The high-quality sequence of complete fosmids can then be tiled into full haplotypes. We demonstrate the method on DNA samples from a number of individuals and fully recover the sequence of both haplotypes from a pool of KIR fosmids. The ability to haplotype and sequence complex immunogenetic regions will bring exciting opportunities to explore the evolution of disease associations of the immune sub-genome. This simple and robust approach can be scaled-up allowing a complex genomic region to be sequenced at a population level. We expect such sequencing to be valuable in disease association research.

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June 1, 2021  |  

Diploid genome assembly and comprehensive haplotype sequence reconstruction

Outside of the simplest cases (haploid, bacteria, or inbreds), genomic information is not carried in a single reference per individual, but rather has higher ploidy (n=>2) for almost all organisms. The existence of two or more highly related sequences within an individual makes it extremely difficult to build high quality, highly contiguous genome assemblies from short DNA fragments. Based on the earlier work on a polyploidy aware assembler, FALCON ( https://github.com/PacificBiosciences/FALCON) , we developed new algorithms and software (“FALCON-unzip”) for de novo haplotype reconstructions from SMRT Sequencing data. We generate two datasets for developing the algorithms and the prototype software: (1) whole genome sequencing data from a highly repetitive diploid fungal (Clavicorona pyxidata) and (2) whole genome sequencing data from an F1 hybrid from two inbred Arabidopsis strains: Cvi-0 and Col-0. For the fungal genome, we achieved an N50 of 1.53 Mb (of the 1n assembly contigs) of the ~42 Mb 1n genome and an N50 of the haplotigs (haplotype specific contigs) of 872 kb from a 95X read length N50 ~16 kb dataset. We found that ~ 45% of the genome was highly heterozygous and ~55% of the genome was highly homozygous. We developed methods to assess the base-level accuracy and local haplotype phasing accuracy of the assembly with short-read data from the Illumina® platform. For the ArabidopsisF1 hybrid genome, we found that 80% of the genome could be separated into haplotigs. The long range accuracy of phasing haplotigs was evaluated by comparing them to the assemblies from the two inbred parental lines. We show that a more complete view of all haplotypes could provide useful biological insights through improved annotation, characterization of heterozygous variants of all sizes, and resolution of differential allele expression. The current Falcon-Unzip method will lead to understand how to solve more difficult polyploid genome assembly problems and improve the computational efficiency for large genome assemblies. Based on this work, we can develop a pipeline enabling routinely assemble diploid or polyploid genomes as haplotigs, representing a comprehensive view of the genomes that can be studied with the information at hand.

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June 1, 2021  |  

Immune regions are no longer incomprehensible with SMRT Sequencing

The complex immune regions of the genome, including MHC and KIR, contain large copy number variants (CNVs), a high density of genes, hyper-polymorphic gene alleles, and conserved extended haplotypes (CEH) with enormous linkage disequilibrium (LDs). This level of complexity and inherent biases of short-read sequencing make it challenging for extracting immune region haplotype information from reference-reliant, shotgun sequencing and GWAS methods. As NGS based genome and exome sequencing and SNP arrays have become a routine for population studies, numerous efforts are being made for developing software to extract and or impute the immune gene information from these datasets. Despite these efforts, the fine mapping of causal variants of immune genes for their well-documented association with cancer, drug-induced hypersensitivity and immune-related diseases, has been slower than expected. This has in many ways limited our understanding of the mechanisms leading to immune disease. In the present work, we demonstrate the advantages of long reads delivered by SMRT Sequencing for assembling complete haplotypes of MHC and KIR gene clusters, as well as calling correct genotypes of genes comprised within them. All the genotype information is detected at allele- level with full phasing information across SNP-poor regions. Genotypes were called correctly from targeted gene amplicons, haplotypes, as well as from a completely assembled 5 Mb contig of the MHC region from a de novo assembly of whole genome shotgun data. De novo analysis pipeline used in all these approaches allowed for reference-free analysis without imputation, a key for interrogation without prior knowledge about ethnic backgrounds. These methods are thus easily adoptable for previously uncharacterized human or non-human species.

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June 1, 2021  |  

Targeted sequencing and chromosomal haplotype assembly using TLA and SMRT Sequencing

With the increasing availability of whole-genome sequencing, haplotype reconstruction of individual genomes, or haplotype assembly, remains unsolved. Like the de novo genome assembly problem, haplotype assembly is greatly simplified by having more long-range information. The Targeted Locus Amplification (TLA) technology from Cergentis has the unique capability of targeting a specific region of the genome using a single primer pair and yielding ~2 kb DNA circles that are comprised of ~500 bp fragments. Fragments from the same circle come from the same haplotype and follow an exponential decay in distance from the target region, with a span that reaches the multi-megabase range. Here, we apply TLA to the BRCA1 gene on NA12878 and then sequence the resulting 2 kb circles on a PacBio RS II. The multiple fragments per circle were iteratively mapped to hg19 and then haplotype assembled using HAPCUT. We show that the 80 kb length of BRCA1 is represented by a single haplotype block, which was validated against GIAB data. We then explored chromosomal-scale haplotype assembly by combining these data with whole genome shotgun PacBio long reads, and demonstrate haplotype blocks approaching the length of chromosome 17 on which BRCA1 lies. Finally, by performing TLA without the amplification step and size selecting for reads >5 kb to maximize the number of fragments per read, we target whole genome haplotype assembly across all chromosomes.

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June 1, 2021  |  

Resolving KIR genotypes and haplotypes simultaneously using Single Molecule, Real-Time Sequencing

The killer immunoglobulin-like receptors (KIR) genes belong to the immunoglobulin superfamily and are widely studied due to the critical role they play in coordinating the innate immune response to infection and disease. Highly accurate, contiguous, long reads, like those generated by SMRT Sequencing, when combined with target-enrichment protocols, provide a straightforward strategy for generating complete de novo assembled KIR haplotypes. We have explored two different methods to capture the KIR region; one applying the use of fosmid clones and one using Nimblegen capture.

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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  |  

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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