Biology subjects

Lewis, A. P.

Publications and source records attributed to Lewis, A. P..

15 recordsLinked to original sources

bioRxiv2025

Long-read sequencing of trios reveals increased germline and postzygotic mutation rates in repetitive DNA

Long-read sequencing (LRS) has improved sensitivity to discover variation in complex repetitive regions, assign parent-of-origin, and distinguish de novo germline from postzygotic mutations (PZMs). Most studies have been limited to population genetic surveys or a few families. We applied three orthogonal sequencing technologies--lIlumina, Oxford Nanopore Technologies, and Pacific Biosciences--to discover and validate de novo mutations (DNMs) in 73 children from 42 autism families (157 individuals). Assaying 2.77 Gbp of the human genome using read-based approaches, we discover on average 95 DNMs per transmission (87.5 de novo single-nucleotide variants and 7.8 indels), including sex chromosomes. We estimate that LRS increases DNM discovery by 20-40% over previous Illumina-based studies of the same families, and more than doubles the discoverable number of PZMs that emerged early in embryonic development. The strict germline mutation rate is 1.30x10-8 substitutions per base pair per generation, strongly driven by the fathers germline (3.95:1), while PZMs increase the rate by 0.23x10-8 with a modest but significant bias toward paternal haplotypes (1.15:1). We show that the mutation rate is significantly increased for classes of repetitive DNA, where segmental duplication (SD) mutation shows a dependence on the length and percent identity of the SD. We find that the mutation rate enrichment in repeats occurs predominantly postzygotically as opposed to in the germline, a likely result of faulty DNA repair and interlocus gene conversion.

genomics↗

bioRxiv2025

Structural variation, selection, and diversification of the NPIP gene family from the human pangenome

The NPIP (nuclear pore interacting protein) gene family has expanded to high copy number in humans and African apes where it has been subject to an excess of amino acid replacement consistent with positive selection (1). Due to the limitations of short-read sequencing, NPIP human genetic diversity has been poorly understood. Using highly accurate assemblies generated from long-read sequencing as part of the human pangenome, we completely characterize 169 human haplotypes (4,665 NPIP paralogs and alleles). Of the 28 NPIP paralogs, just three (NPIPB2, B11, and B14) are fixed at a single copy, and only a single locus, B2, shows no structural variation. Four NPIP paralogs map to large segmental duplication blocks that mediate polymorphic inversions (355 kbp-1.6 Mbp) corresponding to microdeletions associated with developmental delay and autism. Haplotype-based tests of positive selection and selective sweeps identify two paralogs, B9 and B15, within the top percentile for both tests. Using full-length cDNA data from 101 tissue/cell types, we construct paralog-specific gene models and show that 56% (31/55 most abundant isoforms) have not been previously described in RefSeq. We define six distinct translation start sites and other protein structural features that distinguish paralogs, including a variable number tandem repeat that encodes a beta helix of variable size that emerged [~]3.1 million years ago in human evolution. Among the 28 NPIP paralogs, we identify distinct tissue and developmental patterns of expression with only a few maintaining the ancestral testis-enriched expression. A subset of paralogs (NPIPA1, A5, A6-9, B3-5, and B12/B13) show increased brain expression. Our results suggest ongoing positive selection in the human population and rapid diversification of NPIP gene models.

genomics↗

bioRxiv2024

Structural polymorphism and diversity of human segmental duplications

Segmental duplications (SDs) contribute significantly to human disease, evolution, and diversity yet have been difficult to resolve at the sequence level. We present a population genetics survey of SDs by analyzing 170 human genome assemblies where the majority of SDs are fully resolved using long-read sequence assembly. Excluding the acrocentric short arms, we identify 173.2 Mbp of duplicated sequence (47.4 Mbp not present in the telomere-to-telomere reference) distinguishing fixed from structurally polymorphic events. We find that intrachromosomal SDs are among the most variable with rare events mapping near their progenitor sequences. African genomes harbor significantly more intrachromosomal SDs and are more likely to have recently duplicated gene families with higher copy number when compared to non-African samples. A comparison to a resource of 563 million full-length Iso-Seq reads identifies 201 novel, potentially protein-coding genes corresponding to these copy number polymorphic SDs.

genomics↗

bioRxiv2024

Independent expansion, selection and hypervariability of the TBC1D3 gene family in humans

TBC1D3 is a primate-specific gene family that has expanded in the human lineage and has been implicated in neuronal progenitor proliferation and expansion of the frontal cortex. The gene family and its expression have been challenging to investigate because it is embedded in high-identity and highly variable segmental duplications. We sequenced and assembled the gene family using long-read sequencing data from 34 humans and 11 nonhuman primate species. Our analysis shows that this particular gene family has independently duplicated in at least five primate lineages, and the duplicated loci are enriched at sites of large-scale chromosomal rearrangements on chromosome 17. We find that most humans vary along two TBC1D3 clusters where human haplotypes are highly variable in copy number, differing by as many as 20 copies, and structure (structural heterozygosity 90%). We also show evidence of positive selection, as well as a significant change in the predicted human TBC1D3 protein sequence. Lastly, we find that, despite multiple duplications, human TBC1D3 expression is limited to a subset of copies and, most notably, from a single paralog group: TBC1D3-CDKL. These observations may help explain why a gene potentially important in cortical development can be so variable in the human population.

genetics↗

bioRxiv2023

The Complete Sequence and Comparative Analysis of Ape Sex Chromosomes

Apes possess two sex chromosomes--the male-specific Y and the X shared by males and females. The Y chromosome is crucial for male reproduction, with deletions linked to infertility1. The X chromosome carries genes vital for reproduction and cognition2. Variation in mating patterns and brain function among great apes suggests corresponding differences in their sex chromosomes. However, due to their highly repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (chimpanzee, bonobo, gorilla, Bornean and Sumatran orangutans) and a lesser ape, the siamang gibbon. These assemblies allowed us to untangle the intricacies of ape sex chromosome evolution. We found that, compared to the Xs, the ape Ys vary greatly in size and have low alignability and high levels of structural rearrangements. This divergence on the Y arises from the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements, and satellites. Our analysis of Y chromosome genes revealed expansions of multi-copy gene families and signatures of purifying selection. Thus, the Y exhibits dynamic evolution, while the X is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of >100 great ape individuals. These reference assemblies are expected to inform human evolution and conservation genetics of nonhuman apes, all of which are endangered species.

genomics↗

bioRxiv2023

Structurally divergent and recurrently mutated regions of primate genomes

To better understand the pattern of primate genome structural variation, we sequenced and assembled using multiple long-read sequencing technologies the genomes of eight nonhuman primate species, including New World monkeys (owl monkey and marmoset), Old World monkey (macaque), Asian apes (orangutan and gibbon), and African ape lineages (gorilla, bonobo, and chimpanzee). Compared to the human genome, we identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. Across 50 million years of primate evolution, we estimate that 819.47 Mbp or ~27% of the genome has been affected by SVs based on analysis of these primate lineages. We identify 1,607 structurally divergent regions (SDRs) wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (CARDs, ABCD7, OLAH) and new lineage-specific genes are generated (e.g., CKAP2, NEK5) and have become targets of rapid chromosomal diversification and positive selection (e.g., RGPDs). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species for the first time.

genomics↗

bioRxiv2022

Assembly of 43 diverse human Y chromosomes reveals extensive complexity and variation

The prevalence of highly repetitive sequences within the human Y chromosome has led to its incomplete assembly and systematic omission from genomic analyses. Here, we present long-read de novo assemblies of 43 diverse Y chromosomes spanning 180,000 years of human evolution, including two from deep-rooted African Y lineages, and report remarkable complexity and diversity in chromosome size and structure, in contrast with its low level of base substitution variation. The size of the Y chromosome assemblies varies extensively from 45.2 to 84.9 Mbp and include, on average, 81 kbp of novel sequence per Y chromosome. Half of the male-specific euchromatic region is subject to large inversions with a >2-fold higher recurrence rate compared to inversions in the rest of the human genome. Ampliconic sequences associated with these inversions further show differing mutation rates that are sequence context-dependent and some ampliconic genes show evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, the Yq12, is composed of alternating arrays of DYZ1 and DYZ2 repeat units that show extensive variation in the number, size and distribution of these arrays, but retain a 1:1 copy number ratio of the monomer repeats, consistent with the notion that functional or evolutionary forces are acting on this chromosomal region. Finally, our data suggests that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kbp distal to the currently established boundary. The availability of sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of specific traits with Y-chromosomal variants and garnering novel insights into the evolution and function of complex regions of the human genome.

genomics↗

bioRxiv2022

The complete sequence of a human Y chromosome

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure including long palindromes, tandem repeats, and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4, 5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029 base pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, revealing the complete ampliconic structures of TSPY, DAZ, and RBMY gene families; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a prior assembly of the CHM13 genome4 and mapped available population variation, clinical variants, and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

genomics↗

bioRxiv2022

Increased mutation rate and interlocus gene conversion within human segmental duplications.

Single-nucleotide variants (SNVs) within segmental duplications (SDs) have not been systematically assessed because of the difficulty in mapping short-read sequence data to virtually identical repetitive sequences. Using 102 phased human haplotypes, we constructed 1:1 unambiguous alignments spanning high-identity SDs and compared the pattern of SNVs between unique and SD regions. We find that human SNVs are elevated 60% in SDs compared to unique regions. We estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with >7 Mbp of SD sequence converted on average per human haplotype. We develop a genome-wide map of IGC donors and acceptors, including 498 acceptor and 454 donor hotspots affecting the exons of ~800 protein-coding genes. The latter includes 171 genes that have "relocated" on average 1.61 Mbp in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are evolutionarily older when compared to unique sequences with most of this signal originating from putative IGC loci. SNVs within SDs, however, also exhibit a distinct mutational spectrum where there is a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts. In addition, we observe a 7.6% reduction in the frequency of CpG associated mutations when compared to unique DNA. We hypothesize that these distinct mutational properties help to maintain an overall higher GC content of SD DNA when compared to unique DNA, and we show that these GC-favoring mutational events are likely driven by GC-biased conversion between paralogous sequences.

genomics↗

bioRxiv2020

De novo assembly of 64 haplotype-resolved human genomes of diverse ancestry and integrated analysis of structural variation

Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent-child trio data. We present 64 assembled haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average contig N50: 26 Mbp) integrate all forms of genetic variation across even complex loci such as the major histocompatibility complex. We focus on 107,590 structural variants (SVs), of which 68% are inaccessible by short-read sequencing. We identify new SV hotspots (spanning megabases of gene-rich sequence), characterize 130 of the most active mobile element source elements, and find that 63% of all SVs arise by homology-mediated mechanisms--a twofold increase from previous studies. Our resource now enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1,525 expression quantitative trait loci (SV-eQTLs) as well as SV candidates for adaptive selection within the human population.

genetics↗

bioRxiv2020

Targeted long-read sequencing resolves complex structural variants and identifies missing disease-causing variants

BACKGROUNDDespite widespread availability of clinical genetic testing, many individuals with suspected genetic conditions do not have a precise diagnosis. This limits their opportunity to take advantage of state-of-the-art treatments. In such instances, testing sometimes reveals difficult-to-evaluate complex structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in specific genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. METHODSTargeted long-read sequencing (T-LRS) was performed on 33 individuals using Read Until on the Oxford Nanopore platform. This method allowed us to computationally target up to 100 Mbp of sequence per experiment, resulting in an average of 20x coverage of target regions, a 500% increase over background. We analyzed patient DNA for pathogenic substitutions, structural variants, and methylation differences using a single data source. RESULTSThe effectiveness of T-LRS was validated by detecting all genomic aberrations, including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences, previously identified by prior clinical testing. In 6/7 individuals who had complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, which led, in one case, to a change in clinical management. In nine individuals with suspected Mendelian conditions who lacked a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in five and variants of uncertain significance in two others. CONCLUSIONST-LRS can accurately predict pathogenic copy number variants and triplet repeat expansions, resolve complex rearrangements, and identify single-nucleotide variants not detected by other technologies, including short-read sequencing. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority candidate genes and regions or to further evaluate complex clinical testing results. The application of T-LRS will likely increase the diagnostic rate of rare disorders.

genomics↗

bioRxiv2020

Ab-Ligity: Identifying sequence-dissimilar antibodies that bind to the same epitope

Solving the structure of an antibody-antigen complex gives atomic level information of the interactions between an antibody and its antigen, but such structures are expensive and hard to obtain. Alternative experimental sources include epitope mapping and binning experiments which can be used as a surrogate to identify key interacting residues. However, their resolution is usually not sufficient to identify if two antibodies have identical interactions. Computational approaches to this problem have so far been based on the premise that antibodies with similar sequences behave similarly. Such approaches will fail to identify sequence-distant antibodies that target the same epitope. We present Ab-Ligity, a structure-based similarity measure tailored to antibody-antigen interfaces. Using predicted paratopes on model antibody structures, we assessed its ability to identify those antibodies that target highly similar epitopes. Most antibodies adopting similar binding modes can be identified from sequence similarity alone, using methods such as clonotyping. In the challenging subset of antibodies whose sequences differ significantly, Ab-Ligity is still able to predict antibodies that would bind to highly similar epitopes (precision of 0.95 and recall of 0.69). We compared Ab-Ligitys performance to an existing tool for comparing general protein interfaces, InterComp, and showed improved performance on antibody cases alongside a significant speed-up. These results suggest that Ab-Ligity will allow the identification of diverse (sequence-dissimilar) antibodies that bind to the same epitopes from large datasets such as immune repertoires. The tool is available at http://opig.stats.ox.ac.uk/resources.

bioinformatics↗

bioRxiv2020

Evidence of Antibody Repertoire Functional Convergence through Public Baseline and Shared Response Structures

The antibody repertoires of different individuals ought to exhibit significant functional commonality, given that most pathogens trigger a successful immune response in most people. Sequence-based approaches have so far offered little evidence for this phenomenon. For example, a recent study estimated the number of shared ( public) antibody clonotypes in circulating baseline repertoires to be around 0.02% across ten unrelated individuals. However, to engage the same epitope, antibodies only require a similar binding site structure and the presence of key paratope interactions, which can occur even when their sequences are dissimilar. Here, we investigate functional convergence in human antibody repertoires by comparing the anti-body structures they contain. We first structurally profile base-line antibody diversity (using snapshots from 41 unrelated individuals), predicting all modellable distinct structures within each repertoire. This analysis uncovers a high (much greater than random) degree of structural commonality. For instance, around 3% of distinct structures are common to the ten most diverse individual samples ( Public Baseline structures). Our approach is the first computational method to provide support for the long-assumed levels of baseline repertoire functional commonality. We then apply the same structural profiling approach to repertoire snapshots from three individuals before and after flu vaccination, detecting a convergent structural drift indicative of recognising similar epitopes ( Public Response structures). Antibody Model Libraries derived from Public Baseline and Public Response structures represent a powerful geometric basis set of low-immunogenicity candidates exploitable for general or target-focused therapeutic antibody screening.

immunology↗

bioRxiv2019

Thera-SAbDab: the Therapeutic Structural Antibody Database

The Therapeutic Structural Antibody Database (Thera-SAbDab; http://opig.stats.ox.ac.uk/webapps/therasabdab) tracks all antibody- and nanobody-related therapeutics recognised by the World Health Organisation (WHO), and identifies any corresponding structures in the Structural Antibody Database (SAbDab) with near-exact or exact variable domain sequence matches. Thera-SAbDab is synchronised with SAbDab to update weekly, reflecting new Protein Data Bank entries and the availability of new sequence data published by the WHO. Each therapeutic summary page lists structural coverage (with links to the appropriate SAbDab entries), alignments showing where any near-matches deviate in sequence, and accompanying metadata, such as intended target and investigated conditions. Thera-SAbDab can be queried by therapeutic name, by a combination of metadata, or by variable domain sequence - returning all therapeutics that are within a specified sequence identity over a specified region of the query. The sequences of all therapeutics listed in Thera-SAbDab (461 unique molecules, as of 5th August 2019) are downloadable as a single file with accompanying metadata.

immunology↗

bioRxiv2018

The Therapeutic Antibody Profiler (TAP): Five Computational Developability Guidelines

Therapeutic monoclonal antibodies (mAbs) must not only bind to their target but must also be free from 'developability issues', such as poor stability or high levels of aggregation. While small molecule drug discovery benefits from Lipinski's rule of five to guide the selection of molecules with appropriate biophysical properties, there is currently no in silico analog for antibody design. Here, we model the variable domain structures of a large set of post-Phase I clinical-stage antibody therapeutics (CSTs), and calculate an array of metrics to estimate their typical properties. In each case, we contextualize the CST distribution against a snapshot of the human antibody gene repertoire. We describe guideline values for five metrics thought to be implicated in poor developability: the total length of the Complementarity-Determining Regions (CDRs), the extent and magnitude of surface hydrophobicity, positive charge and negative charge in the CDRs, and asymmetry in the net heavy and light chain surface charges. The guideline cut-offs for each property were derived from the values seen in CSTs, and a flagging system is proposed to identify nonconforming candidates. On two mAb drug discovery sets, we were able to selectively highlight sequences with developability issues. We make available the Therapeutic Antibody Profiler (TAP), an open-source computational tool that builds downloadable homology models of variable domain sequences, tests them against our five developability guidelines, and reports potential sequence liabilities and canonical forms. TAP is freely available at http://opig.stats.ox.ac.uk/webapps/sabdab-sabpred/TAP.php.

immunology↗