Learn how Single Molecule, Real-Time (SMRT) Sequencing and the Sequel IIe System and will accelerate your research by delivering highly accurate long reads to provide the most comprehensive view of genomes, transcriptomes and epigenomes.
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.”
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.
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.
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.
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.
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.
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.
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.
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.
Dan Geraghty explains that while there have been decades’ worth of studies associating the genetics of the major histocompatibility complex (MHC), and the highly polymorphic HLA class 1 and 2…
In this webinar, the presenters describe a targeted sequencing workflow that combines Roche NimbleGen’s SeqCap EZ enrichment technology with PacBio’ SMRT Sequencing to provide a more comprehensive view of variants…
In a talk at AGBT 2017, Histogenetics CEO Nezih Cereb reported on how SMRT Sequencing is allowing his team to produce full-length, phased sequences for HLA alleles, which are important…
This tutorial provides an overview of the Long Amplicon Analysis (LAA) application. The LAA algorithm generates highly accurate, phased and full-length consensus sequences from long amplicons. Applications of LAA include…
The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules. KIR genes display abundant copy number variation as well as high levels of polymorphism. As a result, it is challenging to characterize this structurally dynamic region. KIR haplotypes have been analyzed in different species using conventional characterization methods, such as Sanger sequencing and Roche/454 pyrosequencing. However, these methods are time-consuming and often failed to define complete haplotypes, or do not reach allele-level resolution. In addition, most analyses were performed on genomic DNA, and thus were lacking substantial information about transcription and its corresponding modifications. In this paper, we present a single-molecule real-time sequencing approach, using Pacific Biosciences Sequel platform to characterize the KIR transcriptomes in human and rhesus macaque (Macaca mulatta) families. This high-resolution approach allowed the identification of novel Mamu-KIR alleles, the extension of reported allele sequences, and the determination of human and macaque KIR haplotypes. In addition, multiple recombinant KIR genes were discovered, all located on contracted haplotypes, which were likely the result of chromosomal rearrangements. The relatively high number of contracted haplotypes discovered might be indicative of selection on small KIR repertoires and/or novel fusion gene products. This next-generation method provides an improved high-resolution characterization of the KIR cluster in humans and macaques, which eventually may aid in a better understanding and interpretation of KIR allele-associated diseases, as well as the immune response in transplantation and reproduction. Copyright © 2018 by The American Association of Immunologists, Inc.
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