Beyond assembly: the increasing flexibility of single-molecule sequencing technology.

The maturation of high-throughput short-read sequencing technology over the past two decades has shaped the way genomes are studied. Recently, single-molecule, long-read sequencing has emerged as an essential tool in deciphering genome structure and function, including filling gaps in the human reference genome, measuring the epigenome and characterizing splicing variants in the transcriptome. With recent technological developments, these single-molecule technologies have moved beyond genome assembly and are being used in a variety of ways, including to selectively sequence specific loci with long reads, measure chromatin state and protein-DNA binding in order to investigate the dynamics of gene regulation, and rapidly determine copy number variation. These increasingly flexible uses of single-molecule technologies highlight a young and fast-moving part of the field that is leading to a more accessible era of nucleic acid sequencing.

Genomic insights into metabolic flux in hummingbirds.

Hummingbirds are very well adapted to sustain efficient and rapid metabolic shifts. They oxidize ingested nectar to directly fuel flight when foraging but have to switch to oxidizing stored lipids derived from ingested sugars during the night or long-distance migratory flights. Understanding how this organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. To explore these questions, we generated a chromosome-scale genome assembly of the ruby-throated hummingbird (A. colubris) using a combination of long- and short-read sequencing, scaffolding it using existing assemblies. We then used hybrid long- and short-read RNA sequencing of liver and muscle tissue in fasted and fed metabolic states for a comprehensive transcriptome assembly and annotation. Our genomic and transcriptomic data found positive selection of key metabolic genes in nectivorous avian species and deletion of critical genes (SLC2A4, GCK) involved in glucostasis in other vertebrates. We found expression of a fructose-specific version of SLC2A5 putatively in place of insulin-sensitive SLC2A5, with predicted protein models suggesting affinity for both fructose and glucose. Alternative isoforms may even act to sequester fructose to preclude limitations from transport in metabolism. Finally, we identified differentially expressed genes from fasted and fed hummingbirds, suggesting key pathways for the rapid metabolic switch hummingbirds undergo.

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 have not been considered as a feasible replacement for population-scale projects, being a combination of too expensive, not scalable enough or too error-prone. Here we develop an efficient and scalable wet lab and computational protocol, Napu, for Oxford Nanopore Technologies long-read sequencing that seeks to address those limitations. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the National Institutes of Health Center for Alzheimer's and Related Dementias. Using a single PromethION flow cell, we can detect single nucleotide polymorphisms with F1-score comparable to Illumina short-read sequencing. Small indel calling remains difficult within homopolymers and tandem repeats, but achieves good concordance to Illumina indel calls elsewhere. Further, we can discover structural variants with F1-score on par with state-of-the-art de novo assembly methods. Our protocol phases small and structural variants at megabase scales and produces highly accurate, haplotype-specific methylation calls.

Telomere-to-telomere assembly of a complete human X chromosome.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

A Loss of Epigenetic Control Can Promote Cell Death through Reversing the Balance of Pathways in a Signaling Network.

Epigenetic control of regulatory networks is only partially understood. Expression of Insulin-like growth factor-II (IGF2) is controlled by genomic imprinting, mediated by silencing of the maternal allele. Loss of imprinting of IGF2 (LOI) is linked to intestinal and colorectal cancers, causally in murine models and epidemiologically in humans. However, the molecular underpinnings of the LOI phenotype are not clear. Surprisingly, in LOI cells, we find a reversal of the relative activities of two canonical signaling pathways triggered by IGF2, causing further rebalancing between pro- and anti-apoptotic signaling. A predictive mathematical model shows that this network rebalancing quantitatively accounts for the effect of receptor tyrosine kinase inhibition in both WT and LOI cells. This mechanism also quantitatively explains both the stable LOI phenotype and the therapeutic window for selective killing of LOI cells, and thus prevention of epigenetically controlled cancers. These findings suggest a framework for understanding epigenetically modified cell signaling.

Similar evolutionary trajectories in an environmental Cryptococcus neoformans isolate after human and murine infection.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI-SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

Chromosome-specific telomere lengths and the minimal functional telomere revealed by nanopore sequencing.

We developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae Nanopore allows long-read sequencing through the telomere, through the subtelomere, and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end-specific telomere lengths that were stable over 120 cell divisions. These stable chromosome-specific telomere lengths may be explained by slow clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosome end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Y' sequences, tel1Δ rif1Δ double mutants had a very small, but significant, increase in telomere length compared with the tel1Δ single mutant, suggesting an influence of Y' elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2Δ) and found the minimal telomere length to be ∼75 bp. In these est2Δ mutants, there were apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2Δ rad52Δ double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at ∼5 bp per generation. This new method gives quantitative, high-resolution telomere length measurement at each individual chromosome end and suggests possible new biological mechanisms regulating telomere length.

FrameD: framework for DNA-based data storage design, verification, and validation.

MOTIVATION: DNA-based data storage is a quickly growing field that hopes to harness the massive theoretical information density of DNA molecules to produce a competitive next-generation storage medium suitable for archival data. In recent years, many DNA-based storage system designs have been proposed. Given that no common infrastructure exists for simulating these storage systems, comparing many different designs along with many different error models is increasingly difficult. To address this challenge, we introduce FrameD, a simulation infrastructure for DNA storage systems that leverages the underlying modularity of DNA storage system designs to provide a framework to express different designs while being able to reuse common components. RESULTS: We demonstrate the utility of FrameD and the need for a common simulation platform using a case study. Our case study compares designs that utilize strand copies differently, some that align strand copies using multiple sequence alignment algorithms and others that do not. We found that the choice to include multiple sequence alignment in the pipeline is dependent on the error rate and the type of errors being injected and is not always beneficial. In addition to supporting a wide range of designs, FrameD provides the user with transparent parallelism to deal with a large number of reads from sequencing and the need for many fault injection iterations. We believe that FrameD fills a void in the tools publicly available to the DNA storage community by providing a modular and extensible framework with support for massive parallelism. As a result, it will help accelerate the design process of future DNA-based storage systems. AVAILABILITY AND IMPLEMENTATION: The source code for FrameD along with the data generated during the demonstration of FrameD is available in a public Github repository at https://github.com/dna-storage/framed, (https://dx.doi.org/10.5281/zenodo.7757762).

Comparison of red raspberry and wild strawberry fruits reveals mechanisms of fruit type specification.

Belonging to Rosaceae, red raspberry (Rubus idaeus) and wild strawberry (Fragaria vesca) are closely related species with distinct fruit types. While the numerous ovaries become the juicy drupelet fruits in raspberry, their strawberry counterparts become dry and tasteless achenes. In contrast, while the strawberry receptacle, the stem tip, enlarges to become a red fruit, the raspberry receptacle shrinks and dries. The distinct fruit-forming ability of homologous organs in these 2 species allows us to investigate fruit type determination. We assembled and annotated the genome of red raspberry (R. idaeus) and characterized its fruit development morphologically and physiologically. Subsequently, transcriptomes of dissected and staged raspberry fruit tissues were compared to those of strawberry from a prior study. Class B MADS box gene expression was negatively associated with fruit-forming ability, which suggested a conserved inhibitory role of class B heterodimers, PISTILLATA/TM6 or PISTILLATA/APETALA3, for fruit formation. Additionally, the inability of strawberry ovaries to develop into fruit flesh was associated with highly expressed lignification genes and extensive lignification of the ovary pericarp. Finally, coexpressed gene clusters preferentially expressed in the dry strawberry achenes were enriched in "cell wall biosynthesis" and "ABA signaling," while coexpressed clusters preferentially expressed in the fleshy raspberry drupelets were enriched in "protein translation." Our work provides extensive genomic resources as well as several potential mechanisms underlying fruit type specification. These findings provide the framework for understanding the evolution of different fruit types, a defining feature of angiosperms.

The full-length transcriptome of C. elegans using direct RNA sequencing.

Current transcriptome annotations have largely relied on short read lengths intrinsic to the most widely used high-throughput cDNA sequencing technologies. For example, in the annotation of the Caenorhabditis elegans transcriptome, more than half of the transcript isoforms lack full-length support and instead rely on inference from short reads that do not span the full length of the isoform. We applied nanopore-based direct RNA sequencing to characterize the developmental polyadenylated transcriptome of C. elegans Taking advantage of long reads spanning the full length of mRNA transcripts, we provide support for 23,865 splice isoforms across 14,611 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 3452 are novel splice isoforms not present in the WormBase WS265 annotation. Furthermore, we identified 16,342 isoforms in the 3' untranslated region (3' UTR), 2640 of which are novel and do not fall within 10 bp of existing 3'-UTR data sets and annotations. Combining 3' UTRs and splice isoforms, we identified 28,858 full-length transcript isoforms. We also determined that poly(A) tail lengths of transcripts vary across development, as do the strengths of previously reported correlations between poly(A) tail length and expression level, and poly(A) tail length and 3'-UTR length. Finally, we have formatted this data as a publicly accessible track hub, enabling researchers to explore this data set easily in a genome browser.

Comprehensive analysis of structural variants in breast cancer genomes using single-molecule sequencing.

Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of the disease and its progression. We performed whole-genome sequencing of the SKBR3 breast cancer cell line and patient-derived tumor and normal organoids from two breast cancer patients using Illumina/10x Genomics, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT) sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings show that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long reads even at relatively low coverage (25×-30×). Furthermore, we integrated SV and CNV data into a unifying karyotype-graph structure to present a more accurate representation of the mutated cancer genomes. We find hundreds of variants within known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.

Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing.

Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of nucleosome occupancy and methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7 and MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding, and determination of the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate a fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and noncancerous cells.

Author Correction: Nanopore native RNA sequencing of a human poly(A) transcriptome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor.

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.

Chemical and photochemical error rates in light-directed synthesis of complex DNA libraries.

Nucleic acid microarrays are the only tools that can supply very large oligonucleotide libraries, cornerstones of the nascent fields of de novo gene assembly and DNA data storage. Although the chemical synthesis of oligonucleotides is highly developed and robust, it is not error free, requiring the design of methods that can correct or compensate for errors, or select for high-fidelity oligomers. However, outside the realm of array manufacturers, little is known about the sources of errors and their extent. In this study, we look at the error rate of DNA libraries synthesized by photolithography and dissect the proportion of deletion, insertion and substitution errors. We find that the deletion rate is governed by the photolysis yield. We identify the most important substitution error and correlate it to phosphoramidite coupling. Besides synthetic failures originating from the coupling cycle, we uncover the role of imperfections and limitations related to optics, highlight the importance of absorbing UV light to avoid internal reflections and chart the dependence of error rate on both position on the array and position within individual oligonucleotides. Being able to precisely quantify all types of errors will allow for optimal choice of fabrication parameters and array design.

Nanopore native RNA sequencing of a human poly(A) transcriptome.

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.

Epigenetic comparison of CHO hosts and clones reveals divergent methylation and transcription patterns across lineages.

In this study, we examined DNA methylation and transcription profiles of recombinant clones derived from two different Chinese hamster ovary hosts. We found striking epigenetic differences between the clones, with global hypomethylation in the host 1 clones that produce bispecific antibody with higher productivity and complex assembly efficiency. Whereas the methylation patterns were found mostly inherited from the host, the host 1 clones exhibited continued demethylation reflected by the hypomethylation of newly emerged differential methylation regions (DMRs) even at the clone development stage. Several interconnected biological functions and pathways including cell adhesion, regulation of ion transport, and cholesterol biosynthesis were significantly altered between the clones at the RNA expression level and contained DMR in the promoter and/or gene-body of the transcripts, suggesting epigenetic regulation. Indeed, expression changes of epigenetic regulators were observed including writers (Dnmt1, Setdb1), readers (Mecp2), and erasers (Tet3, Kdm3a, Kdm1b/5c) involved in CpG methylation, histone methylation, and heterochromatin maintenance. In addition, we identified putative transcription factors that may be readers or effectors of the epigenetic regulation in these clones. By combining transcriptomics with DNA methylation data, we identified potential processes and factors that may contribute to the variability in cell physiology between different production hosts.

Metagenomic next-generation sequencing of rectal swabs for the surveillance of antimicrobial-resistant organisms on the Illumina Miseq and Oxford MinION platforms.

Antimicrobial resistance (AMR) is a public health threat where efficient surveillance is needed to prevent outbreaks. Existing methods for detection of gastrointestinal colonization of multidrug-resistant organisms (MDRO) are limited to specific organisms or resistance mechanisms. Metagenomic next-generation sequencing (mNGS) is a more rapid and agnostic diagnostic approach for microbiome and resistome investigations. We determined if mNGS can detect MDRO from rectal swabs in concordance with standard microbiology results. We performed and compared mNGS performance on short-read Illumina MiSeq (N = 10) and long-read Nanopore MinION (N = 5) platforms directly from rectal swabs to detect vancomycin-resistant enterococci (VRE) and carbapenem-resistant Gram-negative organisms (CRO). We detected Enterococcus faecium (N = 8) and Enterococcus faecalis (N = 2) with associated van genes (9/10) in concordance with VRE culture-based results. We studied the microbiome and identified CRO, Pseudomonas aeruginosa (N = 1), Enterobacter cloacae (N = 1), and KPC-producing Klebsiella pneumoniae (N = 1). Nanopore real-time analysis detected the blaKPC gene in 2.3 min and provided genetic context (blaKPC harbored on pKPC_Kp46 IncF plasmid). Illumina sequencing provided accurate allelic variant determination (i.e., blaKPC-2) and strain typing of the K. pneumoniae (ST-15). We demonstrated an agnostic approach for surveillance of MDRO, examining advantages of both short- and long-read mNGS methods for AMR detection.

Multi-ancestry fine mapping of interferon lambda and the outcome of acute hepatitis C virus infection.

Clearance of acute infection with hepatitis C virus (HCV) is associated with the chr19q13.13 region containing the rs368234815 (TT/ΔG) polymorphism. We fine-mapped this region to detect possible causal variants that may contribute to HCV clearance. First, we performed sequencing of IFNL1-IFNL4 region in 64 individuals sampled according to rs368234815 genotype: TT/clearance (N = 16) and ΔG/persistent (N = 15) (genotype-outcome concordant) or TT/persistent (N = 19) and ΔG/clearance (N = 14) (discordant). 25 SNPs had a difference in counts of alternative allele >5 between clearance and persistence individuals. Then, we evaluated those markers in an association analysis of HCV clearance conditioning on rs368234815 in two groups of European (692 clearance/1 025 persistence) and African ancestry (320 clearance/1 515 persistence) individuals. 10/25 variants were associated (P < 0.05) in the conditioned analysis leaded by rs4803221 (P value = 4.9 × 10-04) and rs8099917 (P value = 5.5 × 10-04). In the European ancestry group, individuals with the haplotype rs368234815ΔG/rs4803221C were 1.7× more likely to clear than those with the rs368234815ΔG/rs4803221G haplotype (P value = 3.6 × 10-05). For another nearby SNP, the haplotype of rs368234815ΔG/rs8099917T was associated with HCV clearance compared to rs368234815ΔG/rs8099917G (OR: 1.6, P value = 1.8 × 10-04). We identified four possible causal variants: rs368234815, rs12982533, rs10612351 and rs4803221. Our results suggest a main signal of association represented by rs368234815, with contributions from rs4803221, and/or nearby SNPs including rs8099917.

Detecting DNA cytosine methylation using nanopore sequencing.

In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.