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Asset Tag: Variant detection

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

A transcriptome atlas of rabbit revealed by PacBio single-molecule long-read sequencing.

It is widely acknowledged that transcriptional diversity largely contributes to biological regulation in eukaryotes. Since the advent of second-generation sequencing technologies, a large number of RNA sequencing studies have considerably improved our understanding of transcriptome complexity. However, it still remains a huge challenge for obtaining full-length transcripts because of difficulties in the short read-based assembly. In the present study we employ PacBio single-molecule long-read sequencing technology for whole-transcriptome profiling in rabbit (Oryctolagus cuniculus). We totally obtain 36,186 high-confidence transcripts from 14,474 genic loci, among which more than 23% of genic loci and 66% of isoforms have not been annotated yet within the current reference genome. Furthermore, about 17% of transcripts are computationally revealed to be non-coding RNAs. Up to 24,797 alternative splicing (AS) and 11,184 alternative polyadenylation (APA) events are detected within this de novo constructed transcriptome, respectively. The results provide a comprehensive set of reference transcripts and hence contribute to the improved annotation of rabbit genome.

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September 22, 2019

Resistance to ceftazidime-avibactam in Klebsiella pneumoniae due to porin mutations and the increased expression of KPC-3.

We reported the first clinical case of a ceftazidime-avibactam resistant KPC-3-producing Klebsiella pneumoniae (1), from a patient with no history of ceftazidime-avibactam therapy. We now present data documenting mechanisms of ceftazidime-avibactam resistance in this isolate. Whole-genome sequencing (WGS) was performed on two isolates: KP1245 (ceftazidime-avibactam MIC, 4 µg/ml; from blood on hospital day 1; referred to as isolate 1 in our previous report [1]) and KP1244 (ceftazidime-avibactam MIC, 32 µg/ml; from blood on hospital day 2; referred to as isolate 2 in our previous report [2]), using MiSeq (Illumina, San Diego, CA) and PacBio RSII (Menlo Park, CA) systems (2). The in silico multilocus sequence type (ST) was ST258. Single nucleotide polymorphism (SNP) analysis revealed 17 SNPs between KP1245 and KP1244, indicating that the isolates were related but that significant diversity existed in this patient (2). Nonsynonymous mutations are shown in Table 1; the most striking of these is in the OmpK36 porin gene. KP1244 contained a missense mutation predicted to encode a T333N mutation. Both isolates also harbored a mutation predicted to encode R191L in OmpK36 and had a nonfunctional OmpK35, due to a frameshift mutation that truncated the protein at amino acid 42, common to K. pneumoniae ST258 (3). Association between mutations in ompK36 and elevated ceftazidime-avibactam MICs has been shown previously (4). However, T333N, found in one of the ß-sheet domains of the OmpK36 subunit, has not been described in K. pneumoniae; as such, further validation is required to confirm the role of the OmpK36 mutation in this isolate’s ceftazidime-avibactam resistance phenotype.

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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

Predicting an HLA-DPB1 expression marker based on standard DPB1 genotyping: Linkage analysis of over 32,000 samples.

The risk of acute graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation is increased with donor-recipient HLA-DPB1 allele mismatching. The single-nucleotide polymorphism (SNP) rs9277534 within the 3′ untranslated region (UTR) correlates with HLA-DPB1 allotype expression and serves as a marker for permissive HLA-DPB1 mismatches. Since rs9277534 is not routinely typed, we analyzed 32,681 samples of mostly European ancestry to investigate if the rs9277534 allele can be reliably imputed from standard DPB1 genotyping. We confirmed the previously-defined linkages between rs9277534 and 18 DPB1 alleles and established additional linkages for 46 DPB1 alleles. Based on these linkages, the rs9277534 allele could be predicted for 99.6% of the samples based on DPB1 genotypes (99.99% concordance). We demonstrate that 100% prediction accuracy could be achieved if the prediction utilized exon 3 sequence information. DPB1 genotyping based on exon 2 data alone allows no unambiguous rs9277534 allele prediction but was estimated to maintain 99% accuracy for samples of European descent. We conclude that DPB1 genotyping is sufficient to infer the DPB1 expression marker rs9277534 with high accuracy. This information could be used to select donors with permissive HLA-DPB1 mismatches without directly screening for rs9277534. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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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

Conventional and single-molecule targeted sequencing method for specific variant detection in IKBKG while bypassing the IKBKGP1 pseudogene.

In addition to Sanger sequencing, next-generation sequencing of gene panels and exomes has emerged as a standard diagnostic tool in many laboratories. However, these captures can miss regions, have poor efficiency, or capture pseudogenes, which hamper proper diagnoses. One such example is the primary immunodeficiency-associated gene IKBKG. Its pseudogene IKBKGP1 makes traditional capture methods aspecific. We therefore developed a long-range PCR method to efficiently target IKBKG, as well as two associated genes (IRAK4 and MYD88), while bypassing the IKBKGP1 pseudogene. Sequencing accuracy was evaluated using both conventional short-read technology and a newer long-read, single-molecule sequencer. Different mapping and variant calling options were evaluated in their capability to bypass the pseudogene using both sequencing platforms. Based on these evaluations, we determined a robust diagnostic application for unambiguous sequencing and variant calling in IKBKG, IRAK4, and MYD88. This method allows rapid identification of selected primary immunodeficiency diseases in patients suffering from life-threatening invasive pyogenic bacterial infections. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

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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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