: We describe Manta, a method to discover structural variants and indels from next generation sequencing data. Manta is optimized for rapid germline and somatic analysis, calling structural variants, ...medium-sized indels and large insertions on standard compute hardware in less than a tenth of the time that comparable methods require to identify only subsets of these variant types: for example NA12878 at 50× genomic coverage is analyzed in less than 20 min. Manta can discover and score variants based on supporting paired and split-read evidence, with scoring models optimized for germline analysis of diploid individuals and somatic analysis of tumor-normal sample pairs. Call quality is similar to or better than comparable methods, as determined by pedigree consistency of germline calls and comparison of somatic calls to COSMIC database variants. Manta consistently assembles a higher fraction of its calls to base-pair resolution, allowing for improved downstream annotation and analysis of clinical significance. We provide Manta as a community resource to facilitate practical and routine structural variant analysis in clinical and research sequencing scenarios.
Manta is released under the open-source GPLv3 license. Source code, documentation and Linux binaries are available from https://github.com/Illumina/manta.
csaunders@illumina.com
Supplementary data are available at Bioinformatics online.
Whole genome and exome sequencing of matched tumor-normal sample pairs is becoming routine in cancer research. The consequent increased demand for somatic variant analysis of paired samples requires ...methods specialized to model this problem so as to sensitively call variants at any practical level of tumor impurity.
We describe Strelka, a method for somatic SNV and small indel detection from sequencing data of matched tumor-normal samples. The method uses a novel Bayesian approach which represents continuous allele frequencies for both tumor and normal samples, while leveraging the expected genotype structure of the normal. This is achieved by representing the normal sample as a mixture of germline variation with noise, and representing the tumor sample as a mixture of the normal sample with somatic variation. A natural consequence of the model structure is that sensitivity can be maintained at high tumor impurity without requiring purity estimates. We demonstrate that the method has superior accuracy and sensitivity on impure samples compared with approaches based on either diploid genotype likelihoods or general allele-frequency tests.
The Strelka workflow source code is available at ftp://strelka@ftp.illumina.com/.
csaunders@illumina.com
An ultrafast DNA sequence aligner (Isaac Genome Alignment Software) that takes advantage of high-memory hardware (>48 GB) and variant caller (Isaac Variant Caller) have been developed. We demonstrate ...that our combined pipeline (Isaac) is four to five times faster than BWA + GATK on equivalent hardware, with comparable accuracy as measured by trio conflict rates and sensitivity. We further show that Isaac is effective in the detection of disease-causing variants and can easily/economically be run on commodity hardware.
Isaac has an open source license and can be obtained at https://github.com/sequencing.
Abstract
Motivation
In diploid organisms, phasing is the problem of assigning the alleles at heterozygous variants to one of two haplotypes. Reads from PacBio HiFi sequencing provide long, accurate ...observations that can be used as the basis for both calling and phasing variants. HiFi reads also excel at calling larger classes of variation, such as structural or tandem repeat variants. However, current phasing tools typically only phase small variants, leaving larger variants unphased.
Results
We developed HiPhase, a tool that jointly phases SNVs, indels, structural, and tandem repeat variants. The main benefits of HiPhase are (i) dual mode allele assignment for detecting large variants, (ii) a novel application of the A*-algorithm to phasing, and (iii) logic allowing phase blocks to span breaks caused by alignment issues around reference gaps and homozygous deletions. In our assessment, HiPhase produced an average phase block NG50 of 480 kb with 929 switchflip errors and fully phased 93.8% of genes, improving over the current state of the art. Additionally, HiPhase jointly phases SNVs, indels, structural, and tandem repeat variants and includes innate multi-threading, statistics gathering, and concurrent phased alignment output generation.
Availability and implementation
HiPhase is available as source code and a pre-compiled Linux binary with a user guide at https://github.com/PacificBiosciences/HiPhase.
The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the ...only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.
Methods for automated prediction of deleterious protein mutations have utilized both structural and evolutionary information but the relative contribution of these two factors remains unclear. To ...address this, we have used a variety of structural and evolutionary features to create simple deleterious mutation models that have been tested on both experimental mutagenesis and human allele data. We find that the most accurate predictions are obtained using a solvent-accessibility term, the C
β density, and a score derived from homologous sequences, SIFT. A classification tree using these two features has a cross-validated prediction error of 20.5% on an experimental mutagenesis test set when the prior probability for deleterious and neutral cases is equal, whereas this prediction error is 28.8% and 22.2% using either the C
β density or SIFT alone. The improvement imparted by structure increases when fewer homologs are available: when restricted to three homologs the prediction error improves from 26.9% using SIFT alone to 22.4% using SIFT and the C
β density, or 24.8% using SIFT and a noisy C
β density term approximating the inaccuracy of
ab initio structures modeled by the Rosetta method. We conclude that methods for deleterious mutation prediction should include structural information when fewer than five to ten homologs are available, and that
ab initio predicted structures may soon be useful in such cases when high-resolution structures are unavailable.
Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous ...detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30-40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.
Expansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been ...recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads.
We implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR.
Read pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively.
The continuing emergence of viral pathogens and their rapid spread into heavily populated areas around the world underscore the urgency for development of highly effective vaccines to generate ...protective antiviral Ab responses. Many established and newly emerging viral pathogens, including HIV and Ebola viruses, are most prevalent in regions of the world in which
infection remains endemic and vaccination at birth with
bacille Calmette-Guérin (BCG) is widely used. We have investigated the potential for using CD4
T cells arising in response to BCG as a source of help for driving Ab responses against viral vaccines. To test this approach, we designed vaccines comprised of protein immunogens fused to an immunodominant CD4
T cell epitope of the secreted Ag 85B protein of BCG. Proof-of-concept experiments showed that the presence of BCG-specific Th cells in previously BCG-vaccinated mice had a dose-sparing effect for subsequent vaccination with fusion proteins containing the Ag 85B epitope and consistently induced isotype switching to the IgG2c subclass. Studies using an Ebola virus glycoprotein fused to the Ag 85B epitope showed that prior BCG vaccination promoted high-affinity IgG1 responses that neutralized viral infection. The design of fusion protein vaccines with the ability to recruit BCG-specific CD4
Th cells may be a useful and broadly applicable approach to generating improved vaccines against a range of established and newly emergent viral pathogens.
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