Biology subjects

Kolmogorov, M.

Publications and source records attributed to Kolmogorov, M..

At least 19 recordsLinked to original sources

bioRxiv2026

APOBEC3-driven neoantigen-rich cancers co-opt 1q23.3 amplification for tumor-intrinsic immune cloaking

Hypermutational processes, including those driven by the APOBEC3 family of cytidine deaminases, generate abundant neoantigens yet give rise to tumors that evade immune recognition. Here, using multi-omics analyses followed by functional validation, we identified a tumor-intrinsic immune-cloaking mechanism in neoantigen-rich epithelial cancers, characterized by coordinated suppression of antigen presentation, immune-recruiting cytokines and immune-checkpoint programs. In bladder cancer, genome-wide copy-number analysis identified recurrent 1q23.3 amplification as a genomic feature of a neoantigen-high/CD8-low tumor state. Within this locus, NECTIN4 emerged as the dominant candidate effector, outperforming extrachromosomal DNA status as a predictor of immune-neoantigen discordance. Similar associations were observed across breast and lung cancers. Functional studies demonstrated that NECTIN4 was sufficient to establish a T-cell-poor tumor microenvironment and confer resistance to PD-1 blockade in immunocompetent mice. Mechanistically, NECTIN4 engaged a DDR1-SHP2 axis that suppressed STAT1 phosphorylation, silencing tumor-cell immune-engagement programs. NECTIN4 blockade restored STAT1 activity and reduced tumor growth, indicating that the cloaked state is pharmacologically reversible. Mutational signature, breakpoint motif, timing and clonality analyses, together with APOBEC3B expression and germline genetic evidence, linked APOBEC3-mediated mutagenesis to recurrent 1q23.3 amplification encompassing NECTIN4. These findings reveal how neoantigen-generating mutational processes can be coupled to structural genome evolution to enable tumor-intrinsic immune cloaking through a therapeutically targetable NECTIN4-DDR1-SHP2 axis.

cancer biology↗

bioRxiv2026

Chromosome-arm-specific telomere length governs dual modes of structural genome evolution in IDH-mutant astrocytoma

IDH-mutant astrocytomas maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, producing extreme inter-arm telomere length heterogeneity, yet how this heterogeneity shapes structural genome evolution remains unknown. Using Oxford Nanopore long-read sequencing of 20 IDH-mutant astrocytomas, we profiled structural variants (SVs), copy number variants, extrachromosomal DNA (ecDNA) and measured allele-specific telomere lengths from individual long reads. We identified pervasive complex rearrangements, including chromothripsis and foldback events consistent with breakage-fusion-bridge cycles, and widespread ecDNAs. SV breakpoints were enriched at telomeric and centromeric regions regardless of local telomere length, revealing constitutive structural fragility. Arm-level telomere length analysis uncovered a dual-mode model: arms with short telomeres preferentially harbored breakage-associated events, while arms with long ALT-maintained telomeres were enriched for ecDNA and amplification-associated events. These findings identify chromosome-arm-specific telomere length as a determinant of structural genome evolution in ALT-driven tumors.

cancer biology↗

bioRxiv2025

Long-read sequencing of single cell-derived melanoma subclones reveals divergent and parallel genomic and epigenomic evolutionary trajectories

Tumor evolution is driven by various mutational processes, ranging from single-nucleotide vari- ants (SNVs) to large structural variants (SVs) to dynamic shifts in DNA methylation. Current short-read sequencing methods struggle to accurately capture the full spectrum of these genomic and epigenomic alter- ations due to inherent technical limitations. To overcome that, here we introduce an approach for long-read sequencing of single-cell derived subclones, and use it to profile 23 subclones of a mouse melanoma cell line, characterized with distinct growth phenotypes and treatment responses. We develop a computational frame- work for harmonization and joint analysis of different variant types in the evolutionary context. Uniquely, our framework enables detection of recurrent amplifications of putative driver genes, generated by indepen- dent SVs across different lineages, suggesting parallel evolution. In addition, our approach revealed gradual and lineage-specific methylation changes associated with aggressive clonal phenotypes. We also show our set of phylogeny-constrained variant calls along with openly released sequencing data can be a valuable resource for the development of new computational methods.

genomics↗

bioRxiv2025

Diploid genome assembly of human fibroblast cell lines enables clone specific variant calling, improved read mapping and accurate phasing

Human cell lines are fundamental tools in biomedical research and are widely used in disease modeling, drug development, and many other domains. Here, we present chromosome-level, phased diploid genome assemblies of two popular human cell lines: the BJ foreskin fibroblast line and the IMR-90 fetal lung fibroblast line. Our high-quality assemblies, generated using long-read and Hi-C sequencing data, reveal substantial structural variation, including more than 50,000 insertions, deletions, duplications, and inversions compared to the recent T2T-CHM13v2.0 reference. Our assemblies provide detailed maps of genetic variation, enabling more accurate variant calling and the ability to phase reads when using newly generated or historical sequencing data on these cell lines or their derivatives. All assemblies and associated data have been made available as a resource for the research community. We envision that diploid genome assembly will become a cornerstone approach for personalized medicine in the near future.

genomics↗

bioRxiv2024

Long-read sequencing of hundreds of diverse brains provides insight into the impact of structural variation on gene expression and DNA methylation

Structural variants (SVs) drive gene expression in the human brain and are causative of many neurological conditions. However, most existing genetic studies have been based on short-read sequencing methods, which capture fewer than half of the SVs present in any one individual. Long-read sequencing (LRS) enhances our ability to detect disease-associated and functionally relevant structural variants; however, its application in large-scale genomic studies has been limited by challenges in sample preparation and high costs. Here, we leverage a new scalable wet-lab protocol and computational pipeline for whole-genome Oxford Nanopore Technologies sequencing and apply it to neurologically normal control samples from the North American Brain Expression Consortium (NABEC) (European ancestry) and Human Brain Collection Core (HBCC) (African or African admixed ancestry) cohorts. Through this work, we present a publicly available long-read resource from 351 human brain samples (median N50: 27 Kbp and at an average depth of ~40x genome coverage). We discover approximately 234,905 SVs and produce locally phased assemblies that cover 95% of all protein-coding genes in GRCh38. To resolve cis-regulatory effects, we develop ASM-LR, a method for allele-specific methylation analysis from long-read data, revealing both strong and subtle regulatory effects, including numerous novel methylation QTLs masked in unphased models. Our results highlight the power of haplotype-resolved methylation to uncover regulatory mechanisms and establish a foundational resource for exploring how genetic variation shapes gene expression and epigenetic architecture across diverse ancestries.

genomics↗

bioRxiv2024

DeepSomatic: Accurate somatic small variant discovery for multiple sequencing technologies

Somatic variant detection is an integral part of cancer genomics analysis. While most methods have focused on short-read sequencing, long-read technologies now offer potential advantages in terms of repeat mapping and variant phasing. We present DeepSomatic, a deep learning method for detecting somatic SNVs and insertions and deletions (indels) from both short-read and long-read data, with modes for whole-genome and exome sequencing, and able to run on tumor-normal, tumor-only, and with FFPE-prepared samples. To help address the dearth of publicly available training and benchmarking data for somatic variant detection, we generated and make openly available a dataset of five matched tumor-normal cell line pairs sequenced with Illumina, PacBio HiFi, and Oxford Nanopore Technologies, along with benchmark variant sets. Across samples and technologies (short-read and long-read), DeepSomatic consistently outperforms existing callers, particularly for indels.

bioinformatics↗

bioRxiv2023

Personalized Pangenome References

Pangenomes, by including genetic diversity, should reduce reference bias by better representing new samples compared to them. Yet when comparing a new sample to a pangenome, variants in the pangenome that are not part of the sample can be misleading, for example, causing false read mappings. These irrelevant variants are generally rarer in terms of allele frequency, and have previously been dealt with using allele frequency filters. However, this is a blunt heuristic that both fails to remove some irrelevant variants and removes many relevant variants. We propose a new approach, inspired by local ancestry inference methods, that imputes a personalized pangenome subgraph based on sampling local haplotypes according to k-mer counts in the reads. Our approach is tailored for the Giraffe short read aligner, as the indexes it needs for read mapping can be built quickly. We compare the accuracy of our approach to state-of-the-art methods using graphs from the Human Pangenome Reference Consortium. The resulting personalized pangenome pipelines provide faster pangenome read mapping than comparable pipelines that use a linear reference, reduce small variant genotyping errors by 4x relative to the Genome Analysis Toolkit (GATK) best-practice pipeline, and for the first time make short-read structural variant genotyping competitive with long-read discovery methods.

bioinformatics↗

bioRxiv2023

stRainy: assembly-based metagenomic strain phasing using long reads

Bacterial species in microbial communities are often represented by mixtures of strains, distinguished by small variations in their genomes. Despite the functional importance of intra-strain heterogeneity, its characterization from metagenomic sequencing data remains challenging. Short-read approaches can be used to detect small-scale variation between strains, but fail to phase these variants into contiguous haplotypes. Long-read metagenome assemblers can generate contiguous bacterial chromosomes, but often suppress strain-level variation in favor of species-level consensus. Here we present Strainy - an algorithm for strain-level metagenome assembly and phasing from Nanopore and HiFi reads. Strainy takes a de novo metagenomic assembly as input, identifies strain variants which are then phased and assembled into contiguous haplotypes. Using simulated and mock Nanopore and HiFi metagenome data, we show that Strainy assembles accurate and complete strain haplotypes, outperforming current Nanopore-based methods and comparable with HiFi-based algorithms in completeness and accuracy. We then use Strainy to assemble strain haplotypes of a complex environmental metagenome, revealing distinct mutational patterns in bacterial species.

bioinformatics↗

bioRxiv2023

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation

Long-read sequencing technologies substantially overcome the limitations of short-reads but to date have not been considered as feasible replacement at scale due to a combination of being too expensive, not scalable enough, or too error-prone. Here, we develop an efficient and scalable wet lab and computational protocol for Oxford Nanopore Technologies (ONT) long-read sequencing that seeks to provide a genuine alternative to short-reads for large-scale genomics projects. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the NIH Center for Alzheimers and Related Dementias (CARD). Using a single PromethION flow cell, we can detect SNPs with F1-score better than Illumina short-read sequencing. Small indel calling remains difficult within homopolymers and tandem repeats, but is comparable to Illumina calls elsewhere. Further, we can discover structural variants with F1-score comparable to state-of-the-art methods involving Pacific Biosciences HiFi sequencing and trio information (but at a lower cost and greater throughput). Using ONT-based phasing, we can then combine and phase small and structural variants at megabase scales. Our protocol also produces highly accurate, haplotype-specific methylation calls. Overall, this makes large-scale long-read sequencing projects feasible; the protocol is currently being used to sequence thousands of brain-based genomes as a part of the NIH CARD initiative. We provide the protocol and software as open-source integrated pipelines for generating phased variant calls and assemblies.

bioinformatics↗

bioRxiv2022

Automated assembly of high-quality diploid human reference genomes

The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has greatly benefited society1, 2. However, it still has many gaps and errors, and does not represent a biological human genome since it is a blend of multiple individuals3, 4. Recently, a high-quality telomere-to-telomere reference genome, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a duplicate genome, and is thus nearly homozygous5. To address these limitations, the Human Pangenome Reference Consortium (HPRC) recently formed with the goal of creating a collection of high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and automated assembly approaches yields the most complete, accurate, and cost-effective diploid genome assemblies with minimal manual curation. Approaches that used highly accurate long reads and parent-child data to sort haplotypes during assembly outperformed those that did not. Developing a combination of all the top performing methods, we generated our first high- quality diploid reference assembly, containing only [~]4 gaps (range 0-12) per chromosome, most within + 1% of CHM13s length. Nearly 1/4th of protein coding genes have synonymous amino acid changes between haplotypes, and centromeric regions showed the highest density of variation. Our findings serve as a foundation for assembling near-complete diploid human genomes at the scale required for constructing a human pangenome reference that captures all genetic variation from single nucleotides to large structural rearrangements.

genomics↗

bioRxiv2021

Critical Assessment of Metagenome Interpretation - the second round of challenges

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the community-driven initiative for the Critical Assessment of Metagenome Interpretation (CAMI). In its second challenge, CAMI engaged the community to assess their methods on realistic and complex metagenomic datasets with long and short reads, created from [~]1,700 novel and known microbial genomes, as well as [~]600 novel plasmids and viruses. Altogether 5,002 results by 76 program versions were analyzed, representing a 22x increase in results. Substantial improvements were seen in metagenome assembly, some due to using long-read data. The presence of related strains still was challenging for assembly and genome binning, as was assembly quality for the latter. Taxon profilers demonstrated a marked maturation, with taxon profilers and binners excelling at higher bacterial taxonomic ranks, but underperforming for viruses and archaea. Assessment of clinical pathogen detection techniques revealed a need to improve reproducibility. Analysis of program runtimes and memory usage identified highly efficient programs, including some top performers with other metrics. The CAMI II results identify current challenges, but also guide researchers in selecting methods for specific analyses.

bioinformatics↗

bioRxiv2021

Generation of lineage-resolved complete metagenome-assembled genomes by precision phasing

Microbial communities in many environments include distinct lineages of closely related organisms which have proved challenging to separate in metagenomic assembly, preventing generation of complete metagenome-assembled genomes (MAGs). The advent of long and accurate HiFi reads presents a possible means to address this challenge by generating complete MAGs for nearly all sufficiently abundant bacterial genomes in a microbial community. We present a metagenomic HiFi assembly of a complex microbial community from sheep fecal material that resulted in 428 high-quality MAGs from a single sample, the highest resolution achieved with metagenomic deconvolution to date. We applied a computational approach to separate distinct haplotype lineages and identified haplotypes of hundreds of variants across hundreds of kilobases of genomic sequence. Analysis of these haplotypes revealed 220 lineage-resolved complete MAGs, including 44 in single circular contigs, and demonstrated improvement in overall assembly compared to error-prone long reads. We report the characterization of multiple, closely-related microbes within a sample with potential to improve precision in assigning mobile genetic elements to host genomes within complex microbial communities.

microbiology↗

bioRxiv2020

Assembling Long Accurate Reads Using de Bruijn Graphs

Although most existing genome assemblers are based on the de Bruijn graphs, it remains unclear how to construct these graphs for large genomes and large k-mer sizes. This algorithmic challenge has become particularly important with the emergence of long high-fidelity (HiFi) reads that were recently utilized to generate a semi-manual telomere-to-telomere assembly of the human genome and to get a glimpse into biomedically important regions that evaded all previous attempts to sequence them. To enable automated assemblies of long and accurate reads, we developed a fast LJA algorithm that reduces the error rate in these reads by three orders of magnitude (making them nearly error-free) and constructs the de Bruijn graph for large genomes and large k-mer sizes. Since the de Bruijn graph constructed for a fixed k-mer size is typically either too tangled or too fragmented, LJA uses a new concept of a multiplex de Bruijn graph with varying k-mer sizes. We demonstrate that LJA improves on the state-of-the-art assemblers with respect to both accuracy and contiguity and enables automated telomere-to-telomere assemblies of entire human chromosomes.

bioinformatics↗

bioRxiv2019

metaFlye: scalable long-read metagenome assembly using repeat graphs

Long-read sequencing technologies substantially improved assemblies of many isolate bacterial genomes as compared to fragmented assemblies produced with short-read technologies. However, assembling complex metagenomic datasets remains a challenge even for the state-of-the-art long-read assemblers. To address this gap, we present the metaFlye assembler and demonstrate that it generates highly contiguous and accurate metagenome assemblies. In contrast to short-read metagenomics assemblers that typically fail to reconstruct full-length 16S RNA genes, metaFlye captures many 16S RNA genes within long contigs, thus providing new opportunities for analyzing the microbial \"dark matter of life\". We also demonstrate that long-read metagenome assemblers significantly improve full-length plasmid and virus reconstruction as compared to short-read assemblers and reveal many novel plasmids and viruses.

bioinformatics↗

bioRxiv2018

Multiple laboratory mouse reference genomes define strain specific haplotypes and novel functional loci

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like-/- mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

genomics↗

bioRxiv2018

Culture-free generation of microbial genomes from human and marine microbiomes

Our understanding of natural microbial communities is shaped by the careful investigation of a relatively small number of isolated and cultured organisms, and by analysis of genomic sequences obtained by culture-free metagenomic sequencing approaches. Metagenomic shotgun sequencing has facilitated partial reconstruction of strain-level community structure and functional repertoire. Unfortunately, it remains difficult to cost-effectively produce high quality genome drafts for individual microbes without isolation and culture. Recent molecular techniques that partition long DNA fragments and then barcode short fragments derived from them produce \"read clouds\", which are short-read sequences containing long-range information. Here, we present a novel application of a read cloud technique to microbiome samples, as well as Athena, a de novo assembler that uses these barcodes to produce improved metagenomic assemblies. We apply our approach to sequence human stool samples from two healthy individuals, and compare it to existing short read and synthetic long read metagenomic sequencing approaches. We find that read cloud metagenomic sequencing and Athena assembly produce the most complete individual genome drafts. These genome drafts are also highly contiguous (>200kb N50, <10 contigs), even for bacteria that have relatively low (20x) raw short read sequence coverage. We also apply this approach to a significantly more complex marine sediment sample and obtain 23 genome drafts with valuable 16S ribosomal RNA taxonomic marker sequences, nine of which are complete genome drafts. Read cloud metagenomic sequencing allows culture-free generation of high quality microbial genome drafts using only a single shotgun experiment.

genomics↗

bioRxiv2018

Assembly of Long Error-Prone Reads Using Repeat Graphs

The problem of genome assembly is ultimately linked to the problem of the characterization of all repeat families in a genome as a repeat graph. The key reason the de Bruijn graph emerged as a popular short read assembly approach is because it offered an elegant representation of all repeats in a genome that reveals their mosaic structure. However, most algorithms for assembling long error-prone reads use an alternative overlap-layout-consensus (OLC) approach that does not provide a repeat characterization. We present the Flye algorithm for constructing the A-Bruijn (assembly) graph from long error-prone reads, that, in contrast to the k-mer-based de Bruijn graph, assembles genomes using an alignment-based A-Bruijn graph. In difference from existing assemblers, Flye does not attempt to construct accurate contigs (at least at the initial assembly stage) but instead simply generates arbitrary paths in the (unknown) assembly graph and further constructs an assembly graph from these paths. Counter-intuitively, this fast but seemingly reckless approach results in the same graph as the assembly graph constructed from accurate contigs. Flye constructs (overlapping) contigs with possible assembly errors at the initial stage, combines them into an accurate assembly graph, resolves repeats in the assembly graph using small variations between various repeat instances that were left unresolved during the initial assembly stage, constructs a new, less tangled assembly graph based on resolved repeats, and finally outputs accurate contigs as paths in this graph. We benchmark Flye against several state-of-the-art Single Molecule Sequencing assemblers and demonstrate that it generates better or comparable assemblies for all analyzed datasets.

bioinformatics↗

bioRxiv2017

Repeat associated mechanisms of genome evolution and function revealed by the Mus caroli and Mus pahari genomes

Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 to 6 MYA, but that are absent in the Hominidae. In fact, Hominidae show between four-and seven-fold lower rates of nucleotide change and feature turnover in both neutral and functional sequences suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. For example, recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli. This process resulted in thousands of novel, species-specific CTCF binding sites. Our results demonstrate that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.

genomics↗