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We present long-read Tet-assisted pyridine borane sequencing (lrTAPS) for targeted base-resolution sequencing of DNA methylation and hydroxymethylation in regions up to 10 kb from nanogram-level input. Compatible with both Oxford Nanopore and PacBio Single-Molecule Real-Time (SMRT) sequencing, lrTAPS detects methylation with accuracy comparable to short-read Illumina sequencing but with long-range epigenetic phasing. We applied lrTAPS to sequence difficult-to-map regions in mouse embryonic stem cells and to identify distinct methylation events in the integrated hepatitis B virus genome. BackgroundRecent advances in third-generation sequencing methods, including PacBio SMRT sequencing [1-3] and Oxford Nanopore sequencing [4], have enabled long-read and single-molecule sequencing that is distinct from the mainstream short-read Illumina sequencing. These newer sequencing platforms allow unambiguous mapping of repetitive and complex regions of the genome and provide unprecedented opportunities for detecting structural variants, phasing haplotypes, and assembling genomes [5,6]. While Nanopore sequencing still has a high error rate (~10%), the latest SMRT sequencing provides accuracy similar to Illumina sequencing (99.8%) but with an average read length of 13.5 kilobase (kb) compared to~0.3 kb with Illumina [3].Long-read sequencing of DNA modifications, particularly the two abundant modifications-5-methylcytosine (5mC) and 5-hydroxymethylation (5hmC) [7,8], is needed to obtain phased epigenomes that will enable new understanding of the functions of epigenetic modifications, for example allele-specific methylation in genomic imprinting [9] and heterogeneous cancer samples, and diagnosis of brain tumors [10]. Although the SMRT and Nanopore platforms can detect DNA modifications directly, there are major barriers to their application. SMRT sequencing can directly detect DNA modifications using polymerase kinetics information, but requires a minimum of 250× per strand coverage to detect 5mC [11], largely defeating the purpose of long-read sequencing. Several computational methods have been developed to detect base modifications directly from Oxford Nanopore sequencing [12][13][14][15]. However, these approaches require complicated training data from control DNA samples of known methylation status and sophisticated computational analysis, limiting their accuracy to determine 5mC. Moreover, both native SMRT and Oxford Nanopore DNA methylation sequencing require microgram levels of native, unamplified DNA as input. Since

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete understanding of potentially druggable immune mediators of disease. To advance this, we present a comprehensive multi-omic blood atlas in patients with varying COVID-19 severity and compare with influenza, sepsis and healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity revealed cells, their inflammatory mediators and networks as potential therapeutic targets, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Tensor and matrix decomposition of the overall dataset revealed feature groupings linked with disease severity and specificity. Our systems-based integrative approach and blood atlas will inform future drug development, clinical trial design and personalised medicine approaches for COVID-19.