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Asset Tag: Structural variant

September 22, 2019  |  

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ~360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ~25-35 Mya and CFHR1 and CFHR3 ~7-13 Mya). Remarkably, all evolutionary breakpoints share a common ~4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.

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September 22, 2019  |  

The industrial melanism mutation in British peppered moths is a transposable element.

Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of ‘jumping genes’ as a source of major phenotypic novelty.

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September 22, 2019  |  

Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line.

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged, although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available, with nearly 20,000 variants present, most of which were missed by short-read sequencing. Surrounding the important ERBB2 oncogene (also known as HER2), we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the genome and sheds new light on the complex mechanisms involved in cancer genome evolution.© 2018 Nattestad et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019  |  

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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September 22, 2019  |  

Long-read isoform sequencing reveals a hidden complexity of the transcriptional landscape of Herpes Simplex Virus Type 1.

In this study, we used the amplified isoform sequencing technique from Pacific Biosciences to characterize the poly(A)(+) fraction of the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). Our analysis detected 34 formerly unidentified protein-coding genes, 10 non-coding RNAs, as well as 17 polycistronic and complex transcripts. This work also led us to identify many transcript isoforms, including 13 splice and 68 transcript end variants, as well as several transcript overlaps. Additionally, we determined previously unascertained transcriptional start and polyadenylation sites. We analyzed the transcriptional activity from the complementary DNA strand in five convergent HSV gene pairs with quantitative RT-PCR and detected antisense RNAs in each gene. This part of the study revealed an inverse correlation between the expressions of convergent partners. Our work adds new insights for understanding the complexity of the pervasive transcriptional overlaps by suggesting that there is a crosstalk between adjacent and distal genes through interaction between their transcription apparatuses. We also identified transcripts overlapping the HSV replication origins, which may indicate an interplay between the transcription and replication machineries. The relative abundance of HSV-1 transcripts has also been established by using a novel method based on the calculation of sequencing reads for the analysis.

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September 22, 2019  |  

Somatic mosaicism of an intragenic FANCB duplication in both fibroblast and peripheral blood cells observed in a Fanconi anemia patient leads to milder phenotype.

Fanconi anemia (FA) is a rare disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer. Patients harboring X-linked FANCB pathogenic variants usually present with severe congenital malformations resembling VACTERL syndrome with hydrocephalus.We employed the diepoxybutane (DEB) test for FA diagnosis, arrayCGH for detection of duplication, targeted capture and next-gen sequencing for defining the duplication breakpoint, PacBio sequencing of full-length FANCB aberrant transcript, FANCD2 ubiquitination and foci formation assays for the evaluation of FANCB protein function by viral transduction of FANCB-null cells with lentiviral FANCB WT and mutant expression constructs, and droplet digital PCR for quantitation of the duplication in the genomic DNA and cDNA.We describe here an FA-B patient with a mild phenotype. The DEB diagnostic test for FA revealed somatic mosaicism. We identified a 9154 bp intragenic duplication in FANCB, covering the first coding exon 3 and the flanking regions. A four bp homology (GTAG) present at both ends of the breakpoint is consistent with microhomology-mediated duplication mechanism. The duplicated allele gives rise to an aberrant transcript containing exon 3 duplication, predicted to introduce a stop codon in FANCB protein (p.A319*). Duplication levels in the peripheral blood DNA declined from 93% to 7.9% in the span of eleven years. Moreover, the patient fibroblasts have shown 8% of wild-type (WT) allele and his carrier mother showed higher than expected levels of WT allele (79% vs. 50%) in peripheral blood, suggesting that the duplication was highly unstable.Unlike sequence point variants, intragenic duplications are difficult to precisely define, accurately quantify, and may be very unstable, challenging the proper diagnosis. The reversion of genomic duplication to the WT allele results in somatic mosaicism and may explain the relatively milder phenotype displayed by the FA-B patient described here.© 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

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September 22, 2019  |  

Transcriptional fates of human-specific segmental duplications in brain.

Despite the importance of duplicate genes for evolutionary adaptation, accurate gene annotation is often incomplete, incorrect, or lacking in regions of segmental duplication. We developed an approach combining long-read sequencing and hybridization capture to yield full-length transcript information and confidently distinguish between nearly identical genes/paralogs. We used biotinylated probes to enrich for full-length cDNA from duplicated regions, which were then amplified, size-fractionated, and sequenced using single-molecule, long-read sequencing technology, permitting us to distinguish between highly identical genes by virtue of multiple paralogous sequence variants. We examined 19 gene families as expressed in developing and adult human brain, selected for their high sequence identity (average >99%) and overlap with human-specific segmental duplications (SDs). We characterized the transcriptional differences between related paralogs to better understand the birth-death process of duplicate genes and particularly how the process leads to gene innovation. In 48% of the cases, we find that the expressed duplicates have changed substantially from their ancestral models due to novel sites of transcription initiation, splicing, and polyadenylation, as well as fusion transcripts that connect duplication-derived exons with neighboring genes. We detect unannotated open reading frames in genes currently annotated as pseudogenes, while relegating other duplicates to nonfunctional status. Our method significantly improves gene annotation, specifically defining full-length transcripts, isoforms, and open reading frames for new genes in highly identical SDs. The approach will be more broadly applicable to genes in structurally complex regions of other genomes where the duplication process creates novel genes important for adaptive traits.© 2018 Dougherty et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019  |  

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

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September 22, 2019  |  

Long-read sequencing and de novo assembly of a Chinese genome.

Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93?Gb (contig N50: 8.3?Mb, scaffold N50: 22.0?Mb, including 39.3?Mb N-bases), together with 206?Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8?Mb of HX1-specific sequences, including 4.1?Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations.

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September 22, 2019  |  

Long reads: their purpose and place.

In recent years long-read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Analogous to next generation sequencing, the cost of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterizing genomes at high resolution. In this article, we will endeavour to present an introduction to long-read technologies showing: what long reads are; how they are distinct from short reads; why long reads are useful and how they are being used. We will highlight the recent developments in this field, and the applications and potential of these technologies in medical research, and clinical diagnostics and therapeutics.

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September 22, 2019  |  

Jointly aligning a group of DNA reads improves accuracy of identifying large deletions.

Performing sequence alignment to identify structural variants, such as large deletions, from genome sequencing data is a fundamental task, but current methods are far from perfect. The current practice is to independently align each DNA read to a reference genome. We show that the propensity of genomic rearrangements to accumulate in repeat-rich regions imposes severe ambiguities in these alignments, and consequently on the variant calls-with current read lengths, this affects more than one third of known large deletions in the C. Venter genome. We present a method to jointly align reads to a genome, whereby alignment ambiguity of one read can be disambiguated by other reads. We show this leads to a significant improvement in the accuracy of identifying large deletions (=20 bases), while imposing minimal computational overhead and maintaining an overall running time that is at par with current tools. A software implementation is available as an open-source Python program called JRA at https://bitbucket.org/jointreadalignment/jra-src.

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September 22, 2019  |  

A survey of localized sequence rearrangements in human DNA.

Genomes mutate and evolve in ways simple (substitution or deletion of bases) and complex (e.g. chromosome shattering). We do not fully understand what types of complex mutation occur, and we cannot routinely characterize arbitrarily-complex mutations in a high-throughput, genome-wide manner. Long-read DNA sequencing methods (e.g. PacBio, nanopore) are promising for this task, because one read may encompass a whole complex mutation. We describe an analysis pipeline to characterize arbitrarily-complex ‘local’ mutations, i.e. intrachromosomal mutations encompassed by one DNA read. We apply it to nanopore and PacBio reads from one human cell line (NA12878), and survey sequence rearrangements, both real and artifactual. Almost all the real rearrangements belong to recurring patterns or motifs: the most common is tandem multiplication (e.g. heptuplication), but there are also complex patterns such as localized shattering, which resembles DNA damage by radiation. Gene conversions are identified, including one between hemoglobin gamma genes. This study demonstrates a way to find intricate rearrangements with any number of duplications, deletions, and repositionings. It demonstrates a probability-based method to resolve ambiguous rearrangements involving highly similar sequences, as occurs in gene conversion. We present a catalog of local rearrangements in one human cell line, and show which rearrangement patterns occur.

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September 22, 2019  |  

Reproducible integration of multiple sequencing datasets to form high-confidence SNP, indel, and reference calls for five human genome reference materials

Benchmark small variant calls from the Genome in a Bottle Consortium (GIAB) for the CEPH/HapMap genome NA12878 (HG001) have been used extensively for developing, optimizing, and demonstrating performance of sequencing and bioinformatics methods. Here, we develop a reproducible, cloud-based pipeline to integrate multiple sequencing datasets and form benchmark calls, enabling application to arbitrary human genomes. We use these reproducible methods to form high-confidence calls with respect to GRCh37 and GRCh38 for HG001 and 4 additional broadly-consented genomes from the Personal Genome Project that are available as NIST Reference Materials. These new genomes’ broad, open consent with few restrictions on availability of samples and data is enabling a uniquely diverse array of applications. Our new methods produce 17% more high-confidence SNPs, 176% more indels, and 12% larger regions than our previously published calls. To demonstrate that these calls can be used for accurate benchmarking, we compare other high-quality callsets to ours (e.g., Illumina Platinum Genomes), and we demonstrate that the majority of discordant calls are errors in the other callsets, We also highlight challenges in interpreting performance metrics when benchmarking against imperfect high-confidence calls. We show that benchmarking tools from the Global Alliance for Genomics and Health can be used with our calls to stratify performance metrics by variant type and genome context and elucidate strengths and weaknesses of a method.

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September 22, 2019  |  

IMSindel: An accurate intermediate-size indel detection tool incorporating de novo assembly and gapped global-local alignment with split read analysis.

Insertions and deletions (indels) have been implicated in dozens of human diseases through the radical alteration of gene function by short frameshift indels as well as long indels. However, the accurate detection of these indels from next-generation sequencing data is still challenging. This is particularly true for intermediate-size indels (=50?bp), due to the short DNA sequencing reads. Here, we developed a new method that predicts intermediate-size indels using BWA soft-clipped fragments (unmatched fragments in partially mapped reads) and unmapped reads. We report the performance comparison of our method, GATK, PINDEL and ScanIndel, using whole exome sequencing data from the same samples. False positive and false negative counts were determined through Sanger sequencing of all predicted indels across these four methods. The harmonic mean of the recall and precision, F-measure, was used to measure the performance of each method. Our method achieved the highest F-measure of 0.84 in one sample, compared to 0.56 for GATK, 0.52 for PINDEL and 0.46 for ScanIndel. Similar results were obtained in additional samples, demonstrating that our method was superior to the other methods for detecting intermediate-size indels. We believe that this methodology will contribute to the discovery of intermediate-size indels associated with human disease.

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