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Key Points There are two major paradigms in next-generation sequencing (NGS) technology: short-read sequencing and long-read sequencing. Short-read sequencing approaches provide lower-cost, higher-accuracy data that are useful for population-level research and clinical variant discovery. By contrast, long-read approaches provide read lengths that are well suited for de novo genome assembly applications and full-length isoform sequencing. NGS technologies have been evolving over the past 10 years, leading to substantial improvements in quality and yield; however, certain approaches have proven to be more effective and adaptable than others. Recent improvements in chemistry, costs, throughput and accessibility are driving the emergence of new, varied technologies to address applications that were not previously possible. These include integrated long-read and short-read sequencing studies, routine clinical DNA sequencing, real-time pathogen DNA monitoring and massive population-level projects. Although massive strides are being made in this technology, several notable limitations remain. The time required to sequence and analyse data limits the use of NGS in clinical applications in which time is an important factor; the costs and error rates of long-read sequencing make it prohibitive for routine use, and ethical considerations can limit the public and private use of genetic data. We can expect increasing democratization and options for NGS in the future. Many new instruments with varied chemistries and applications are being released or being developed. Advances in DNA sequencing technologies have led to vast increases in the diversity of sequencing-based applications and in the amount of data generated. This Review discusses the current state-of-the-art technologies in both short-read and long-read DNA sequencing, their underlying mechanisms, relative strengths and limitations, and emerging applications. Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alternatively, other approaches now aim to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions. These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.

We present RaGOO, a reference-guided contig ordering and orienting tool that leverages the speed and sensitivity of Minimap2 to accurately achieve chromosome-scale assemblies in minutes. After the pseudomolecules are constructed, RaGOO identifies structural variants, including those spanning sequencing gaps. We show that RaGOO accurately orders and orients 3 de novo tomato genome assemblies, including the widely used M82 reference cultivar. We then demonstrate the scalability and utility of RaGOO with a pan-genome analysis of 103 Arabidopsis thaliana accessions by examining the structural variants detected in the newly assembled pseudomolecules. RaGOO is available open source at https://github.com/malonge/RaGOO .

Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but also by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mycobacterium tuberculosis ( Mtb ) genes. We explored the role of codon pair bias by recoding Mtb genes ( rpoB, mmpL3 , and ndh ) and assessing their expression in the closely related and tractable model organism M. smegmatis . To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which, in turn, led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation. Mycobacterium tuberculosis ( Mtb ) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb . We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.

Given that early-stage oral cavity squamous cell carcinoma (OCSCC) has a high propensity for subclinical nodal metastasis, elective neck dissection has become standard practice for many patients with clinically negative nodes. Unfortunately, for most patients without regional metastasis, this risk-averse treatment paradigm results in unnecessary morbidity. To develop and validate predictive models of occult nodal metastasis from clinicopathological variables that were available after surgical extirpation of the primary tumor and to compare predictive performance against depth of invasion (DOI), the currently accepted standard. This diagnostic modeling study collected clinicopathological variables retrospectively from 7 tertiary care academic medical centers across the US. Participants included adult patients with early-stage OCSCC without nodal involvement who underwent primary surgical extirpation with or without upfront elective neck dissection. These patients were initially evaluated between January 1, 2000, and December 31, 2019. Largest tumor dimension, tumor thickness, DOI, margin status, lymphovascular invasion, perineural invasion, muscle invasion, submucosal invasion, dysplasia, histological grade, anatomical subsite, age, sex, smoking history, race and ethnicity, and body mass index (calculated as weight in kilograms divided by height in meters squared). Occult nodal metastasis identified either at the time of elective neck dissection or regional recurrence within 2 years of initial surgery. Of the 634 included patients (mean [SD] age, 61.2 [13.6] years; 344 men [54.3%]), 114 (18.0%) had occult nodal metastasis. Patients with occult nodal metastasis had a higher frequency of lymphovascular invasion (26.3% vs 8.1%; P < .001), perineural invasion (40.4% vs 18.5%; P < .001), and margin involvement by invasive tumor (12.3% vs 6.3%; P = .046) compared with those without pathological lymph node metastasis. In addition, patients with vs those without occult nodal metastasis had a higher frequency of poorly differentiated primary tumor (20.2% vs 6.2%; P < .001) and greater DOI (7.0 vs 5.4 mm; P < .001). A predictive model that was built with XGBoost architecture outperformed the commonly used DOI threshold of 4 mm, achieving an area under the curve of 0.84 (95% CI, 0.80-0.88) vs 0.62 (95% CI, 0.57-0.67) with DOI. This model had a sensitivity of 91.7%, specificity of 72.6%, positive predictive value of 39.3%, and negative predictive value of 97.8%. Results of this study showed that machine learning models that were developed from multi-institutional clinicopathological data have the potential to not only reduce the number of pathologically node-negative neck dissections but also accurately identify patients with early OCSCC who are at highest risk for nodal metastases.

ObjectiveQuinolinic acid (QA), a kynurenine (KYN)/tryptophan (TRP) pathway metabolite, is an N-methyl-D-aspartate receptor agonist that can produce excitotoxic neuron damage. Type I and II interferons (IFNs) stimulate the KYN/TRP pathway, producing elevated QA/kynurenic acid (KA), a potential neurotoxic imbalance that may contribute to SLE-mediated cognitive dysfunction. We determined whether peripheral blood interferon-stimulated gene (ISG) expression associates with elevated serum KYN:TRP and QA:KA ratios in SLE.MethodsISG expression (whole-blood RNA sequencing) and serum metabolite ratios (high-performance liquid chromatography) were measured in 72 subjects with SLE and 73 healthy controls (HCs). ISG were identified from published gene sets and individual IFN scores were derived to analyse associations with metabolite ratios, clinical parameters and neuropsychological assessments. SLE analyses were grouped by level of ISG expression (‘IFN high’, ‘IFN low’ and ‘IFN similar to HC’) and level of monocyte-associated gene expression (using CIBERSORTx).ResultsSerum KYN:TRP and QA:KA ratios were higher in SLE than in HC (p<0.01). 933 genes were differentially expressed ≥2-fold in SLE versus HC (p<0.05). 70 of the top 100 most highly variant genes were ISG. Approximately half of overexpressed genes that correlated with KYN:TRP and QA:KA ratios (p<0.05) were ISG. In 36 IFN-high subjects with SLE, IFN scores correlated with KYN:TRP ratios (p<0.01), but not with QA:KA ratios. Of these 36 subjects, 23 had high monocyte-associated gene expression, and in this subgroup, the IFN scores correlated with both KY:NTRP and QA:KA ratios (p<0.05).ConclusionsHigh ISG expression correlated with elevated KYN:TRP ratios in subjects with SLE, suggesting IFN-mediated KYN/TRP pathway activation, and with QA:KA ratios in a subset with high monocyte-associated gene expression, suggesting that KYN/TRP pathway activation may be particularly important in monocytes. These results need validation, which may aid in determining which patient subset may benefit from therapeutics directed at the IFN or KYN/TRP pathways to ameliorate a potentially neurotoxic QA/KA imbalance.

BackgroundCognitive dysfunction (CD) is highly prevalent in systemic lupus erythematosus (SLE) with significant impact on quality of life, yet SLE-mediated mechanisms for CD remain poorly understood. Quinolinic acid (QA), a metabolite of the kynurenine (KYN)/tryptophan (TRP) pathway, is a N-methyl-D-aspartate receptor (NMDAR) agonist that can cause excessive glutamatergic excitotoxicity to neurons,1 while kynurenic acid (KA) is an NMDAR antagonist with potential to protect neurons from excitotoxic damage (figure 1).1 Type I and II interferon (IFN) contributes to SLE pathogenesis and stimulates the KYN/TRP pathway, producing an elevated QA/KA ratio, a potential neurotoxic imbalance. We determined whether peripheral blood IFN- stimulated gene (ISG) expression associates with elevated serum KYN/TRP and QA/KA ratios in SLE.MethodsWe measured ISG expression (whole blood RNA sequencing) and serum metabolite ratios (High Performance Liquid Chromatography) in 72 SLE subjects and 73 healthy controls (HC). We identified ISG based on published gene sets from Arazi et al2 (“ISG-A,” N=110 ISG), Chiche et al3 (“19 type I ISG” more responsive to type I than type II IFN), and the Interferome database.4 We derived individual IFN scores to analyze associations with metabolite ratios and clinical parameters. These analyses were performed in SLE subgroups based on level of ISG expression (“IFN high”, “IFN low” and “IFN similar to HC”) and, using CIBERSORTx, according to the level of monocyte-associated gene expression.ResultsSerum KYN/TRP and QA/KA ratios were higher in SLE versus HC (p<0.01) (table 1). SLE subjects were racially diverse, reflective of disease demographics, with a wide range of disease activity (SLEDAI scores ranging 0-29) and medication use. There were no demographic differences between SLE and HC. Nine hundred thirty-three genes were differentially expressed ≥2-fold in SLE versus HC, with 762 genes overexpressed and 171 underexpressed (p<0.05). Seventy of the top 100 most highly variant genes were ISG. Of the 762 overexpressed genes in SLE subjects, 144 positively correlated with KYN/TRP ratios (p<0.05) and 71 (49%) of these were ISG. Similarly, 81 of the 762 overexpressed genes positively correlated with QA/KA ratios in SLE subjects (p<0.05), and 38 (47%) of these were ISG. In 36 “IFN high” SLE subjects, IFN scores correlated with KYN/TRP ratios (p<0.01), but not with QA/KA ratios (table 2). Of these 36 “IFN high” SLE subjects, 23 had high monocyte-associated gene expression and in this subgroup, the IFN scores correlated with both KYN/TRP and QA/KA ratios (p<0.05) (table 3).ConclusionsSLE subjects demonstrate increased KYN/TRP pathway metabolite ratios, and high ISG expression correlated with elevated KYN/TRP ratios, suggesting IFN-mediated KYN/TRP pathway activation. High ISG expression also correlated with QA/KA ratios in SLE subjects with high monocyte-associated gene expression, suggesting that KYN/TRP pathway activation may be particularly important in monocytes. These results need validation, which may aid in determining which subset of patients may benefit from therapeutics directed at the IFN or KYN/TRP pathways to ameliorate a potentially neurotoxic QA/KA imbalance.Abstract 621 Figure 1The Kynurenine/Tryptophan Pathway. This is a simplified schematic of the KYN/TRP pathway, highlighting the intermediates and enzymes involved in the production of quinolinic acid (QA) and kynurenic acid (KA). The enzyme IDO is stimulated by inflammatory cytokines, such as IFN, that results in the breakdown of TRP into KYN. KYN may be further metabolized by KMO ultimately to QA, an NMDAR agonist, or by KAT to KA, an NMDAR antagonist. Since the enzyme KMO has higher affinity for KYN than KAT, metabolism proceeds preferentially towards the production of QA in the setting of inflammation.5* IDO, indoleamine 2,3-dioxygenase; IFNα, interferon-alpha; IFNγ, interferon-gamma; NMDAR, N-methyl D-aspartate receptor; TNFα, tumor necrosis factor-alpha.Abstract 621 Table 1SLE and healthy control (HC) subject characteristics and KYN/TRP pathway metabolite ratios. All data is reported either as a mean (or median where indicated) ± standard deviation (or interquartile range), or as a frequency (%). All data refers to that which was collected at the time of evaluation Subject characteristics SLE (N = 72) HC (N = 73) p Age(mean # years ± SD, range) 37.9 ± 9.6(22 – 57) 36.2 ± 9.5(18 – 55) 0.28 Ethnicity (Hispanic/Latino) 13 (18.1%) 13 (17.8%) 0.97 Race Black 43 (59.7%) 41 (56.2%) 0.91 White 16 (22.2%) 18 (24.7%) Other 13 (18.1%) 14 (19.2%) KYN/TRP ratioMedian ± IQR (range) 0.04 ± 0.03 (0.01 - 0.23) 0.03 ± 0.01 (0.01 - 0.13) <0.01 QA/KA ratioMedian ± IQR (range) 18.4 ± 14.7 (4.0 - 121.2) 8.9 ± 5.8 (2.9 - 45.9) <0.01 Disease durationMean ± SD (range) 12.3 ± 8.5(1 – 38) n/a n/a SELENA SLEDAI scoreMean ± SD (range) 5.4 ± 5.1(0 – 29) n/a n/a Prednisone dose(mg/day; median ± IQR, range) 2.5 ± 10.0(0 – 75) n/a n/a Current hydroxychloroquine use 54 (75.0%) n/a n/a Current immunosuppressant use 34 (47.2%) n/a n/a Anti-dsDNA positive (> 29 IU/mL) 50 (69.4%) n/a n/a C3 low (< 81 mg/dL) 28 (38.9%) n/a n/a C4 low (< 13 mg/dL) 24 (33.3%) n/a n/a Abstract 621 Table 2Correlations Between IFN Scores and Serum KYN/TRP Pathway Metabolite Levels in SLE Subjects According to ISG Expression Subgroup. SLE subjects were assigned to 1 of 3 subgroups: “IFN high” (Z-IFN score ≥2 SD above HC mean), “IFN low” (Z-IFN score ≥1 to <2) or “IFN similar to HC” (Z-IFN score <1). In each subgroup, correlations between both IFN scores (derived from ISG-A and the 19 type I ISG) and metabolite ratios are displayed. ISG Expression Subgroup IFN Score Method(z-scores) KYN/TRP (rs) p QA/KA (rs) p IFN high(N=36) ISG-A 0.490 0.002* 0.129 0.453 19 Type I ISG 0.487 0.003* 0.216 0.205 IFN low(N=23) ISG-A -0.174 0.427 -0.286 0.187 19 Type I ISG 0.269 0.215 -0.210 0.335 IFN similar to HC(N=13) ISG-A 0.170 0.578 -0.126 0.681 19 Type I ISG 0.242 0.426 0.000 1.000 * remained significant after Benjamini-Hochberg correction for multiple comparisonsAbstract 621 Table 3Correlations Between IFN Scores and Serum KYN/TRP Pathway Metabolite Levels in SLE Subjects According to ISG and Monocyte-Associated Gene Expression Subgroups. In addition to the ISG expression subgroups previously described in Table 2, SLE subjects were further designated as either “Monocyte High” (monocyte-associated gene expression > HC mean) or “Monocyte Low” (monocyte- associated gene expression ≤ HC mean). In each subgroup, correlations between both IFN scores (derived from ISG-A and the 19 type I ISG) and metabolite ratios are displayed. ISG Expression Subgroup Monocyte- Associated Gene Expression Subgroup IFN Score Method (z-scores) KYN/TRP (rs) p QA/KA (rs) p IFN high(N=36) Monocyte High(N=23) ISG-A 0.504 0.014* 0.429 0.041* 19 Type I ISG 0.532 0.009* 0.419 0.046* Monocyte Low(N=13) ISG-A 0.440 0.133 -0.132 0.668 19 Type I ISG 0.330 0.271 0.044 0.887 IFN low(N=23) Monocyte High(N=17) ISG-A -0.289 0.260 -0.326 0.202 19 Type I ISG 0.336 0.188 -0.252 0.328 Monocyte Low(N=6) ISG-A 0.547 0.261 0.137 0.796 19 Type I ISG 0.429 0.396 0.073 0.890 IFN similar to HC(N=13) Monocyte High(N=8) ISG-A -0.071 0.867 -0.199 0.637 19 Type I ISG 0.048 0.911 -0.024 0.955 Monocyte Low(N=5) ISG-A 0.785 0.116 0.110 0.861 19 Type I ISG 0.500 0.391 0.100 0.873 * remained significant after Benjamini-Hochberg correction for multiple comparisonsReferencesSchwarcz R and Stone TW. The kynurenine pathway and the brain: Challenges, controversies and promises. Neuropharmacology 2017;112:237-247.Arazi A, Rao DA, Berthier CC, et al. The immune cell landscape in kidneys of patients with lupus nephritis. Nat Immunol 2019;20:902-914.Chiche L, Jourde-Chiche N, Whalen E, et al. Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures. Arthritis Rheumatol 2014;66:1583-95.Rusinova I, Forster S, Yu S, et al. Interferome v2.0: an updated database of annotated interferon- regulated genes. Nucleic Acids Res 2013;41:D1040-6.Lugo-Huitron R, Ugalde Muniz P, Pineda B, et al. Quinolinic acid: an endogenous neurotoxin with multiple targets. Oxid Med Cell Longev 2013;2013:104024.

MaizeCODE is a project aimed at identifying and analyzing functional elements in the maize genome. In its initial phase, MaizeCODE assayed up to five tissues from four maize strains (B73, NC350, W22, TIL11) by RNA-Seq, Chip-Seq, RAMPAGE, and small RNA sequencing. To facilitate reproducible science and provide both human and machine access to the MaizeCODE data, we enhanced SciApps, a cloud-based portal, for analysis and distribution of both raw data and analysis results. Based on the SciApps workflow platform, we generated new components to support the complete cycle of MaizeCODE data management. These include publicly accessible scientific workflows for the reproducible and shareable analysis of various functional data, a RESTful API for batch processing and distribution of data and metadata, a searchable data page that lists each MaizeCODE experiment as a reproducible workflow, and integrated JBrowse genome browser tracks linked with workflows and metadata. The SciApps portal is a flexible platform that allows the integration of new analysis tools, workflows, and genomic data from multiple projects. Through metadata and a ready-to-compute cloud-based platform, the portal experience improves access to the MaizeCODE data and facilitates its analysis.

ObjectiveWe assessed whether famotidine improved inflammation and symptomatic recovery in outpatients with mild to moderate COVID-19.DesignRandomised, double-blind, placebo-controlled, fully remote, phase 2 clinical trial (NCT04724720) enrolling symptomatic unvaccinated adult outpatients with confirmed COVID-19 between January 2021 and April 2021 from two US centres. Patients self-administered 80 mg famotidine (n=28) or placebo (n=27) orally three times a day for 14 consecutive days. Endpoints were time to (primary) or rate of (secondary) symptom resolution, and resolution of inflammation (exploratory).ResultsOf 55 patients in the intention-to-treat group (median age 35 years (IQR: 20); 35 women (64%); 18 African American (33%); 14 Hispanic (26%)), 52 (95%) completed the trial, submitting 1358 electronic symptom surveys. Time to symptom resolution was not statistically improved (p=0.4). Rate of symptom resolution was improved for patients taking famotidine (p<0.0001). Estimated 50% reduction of overall baseline symptom scores were achieved at 8.2 days (95% CI: 7 to 9.8 days) for famotidine and 11.4 days (95% CI: 10.3 to 12.6 days) for placebo treated patients. Differences were independent of patient sex, race or ethnicity. Five self-limiting adverse events occurred (famotidine, n=2 (40%); placebo, n=3 (60%)). On day 7, fewer patients on famotidine had detectable interferon alpha plasma levels (p=0.04). Plasma immunoglobulin type G levels to SARS-CoV-2 nucleocapsid core protein were similar between both arms.ConclusionsFamotidine was safe and well tolerated in outpatients with mild to moderate COVID-19. Famotidine led to earlier resolution of symptoms and inflammation without reducing anti-SARS-CoV-2 immunity. Additional randomised trials are required.

Despite significant advances in our understanding of genetic cancer susceptibility, known inherited cancer predisposition syndromes explain at most 20% of early-onset cancers. As early-onset cancer prevalence continues to increase, the need to assess previously inaccessible areas of the human genome, harnessing a trio or quad family-based architecture for variant filtration, may reveal further insights into cancer susceptibility. To assess a broader spectrum of variation than can be ascertained by multi-gene panel sequencing, or even whole genome sequencing with short reads, we employed long read whole genome sequencing using an Oxford Nanopore Technology (ONT) PromethION of 3 families containing an early-onset cancer proband using a trio or quad family architecture. Analysis included 2 early-onset colorectal cancer family trios and one quad consisting of two siblings with testicular cancer, all with unaffected parents. Structural variants (SVs), epigenetic profiles and single nucleotide variants (SNVs) were determined for each individual, and a filtering strategy was employed to refine and prioritize candidate variants based on the family architecture. The family architecture enabled us to focus on inapposite variants while filtering variants shared with the unaffected parents, significantly decreasing background variation that can hamper identification of potentially disease causing differences. Candidate e and compound heterozygous variants were identified in this way. Gene expression, in matched neoplastic and pre-neoplastic lesions, was assessed for one trio. Our study demonstrates the feasibility of a streamlined analysis of genomic variants from long read ONT whole genome sequencing and a way to prioritize key variants for further evaluation of pathogenicity, while revealing what may be missing from panel based analyses.