Conventional targeted sequencing methods eliminate many of the benefits of nanopore sequencing, such as the ability to accurately detect structural variants (SVs) or epigenetic modifications. The ReadUntil method allows nanopore devices to selectively eject reads from pores in real-time, which could enable purely computational targeted sequencing. However this requires rapid identification of on-target reads, and most mapping methods require computationally intensive basecalling. We present UNCALLED ( github.com/skovaka/UNCALLED ), an open-source mapper that rapidly matches streaming nanopore current signals to a reference sequence. UNCALLED probabilistically considers k-mers that the signal could represent, and then prunes the candidates based on the reference encoded within an FM-index. We used UNCALLED to deplete sequencing of known bacterial genomes within a metagenomics community, enriching the remaining species by 4.46 fold. UNCALLED also enriched 148 human genes associated with hereditary cancers to 29.6x coverage using one MinION flowcell, enabling accurate detection of SNPs, indels, SVs, and methylation in these genes.

Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300 bp) of 2nd generation sequencing. Not only do the reads (or paired-end reads) need to straddle a breakpoint, but repetitive elements often lead to ambiguities in the alignment of short reads. We propose to use the long-reads (up to 20 kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Though nanopore sequencing currently has a relatively high mismatch rate that precludes base substitution and small frameshift mutation detection, its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency.We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.

Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. Here, we sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole-genome sequencing in predicting AST results. Using a cohort of 40 clinical isolates (21 carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae, 10 non-carbapenemase-producing carbapenem-resistant K. pneumoniae, and 9 carbapenem-susceptible K. pneumoniae isolates), three separate sequencing and analysis pipelines were performed, as follows: (i) a real-time Nanopore analysis approach identifying acquired AMR genes, (ii) an assembly-based Nanopore approach identifying acquired AMR genes and chromosomal mutations, and (iii) an approach using short-read correction of Nanopore assemblies. The short-read correction of Nanopore assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results. With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 h from subcultured isolates. Assemblies sufficient for full resistance gene and single-nucleotide polymorphism annotation were available within 14 h from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40 K. pneumoniae isolates was 77% (range, 30% to 100%) and 92% (range, 80% to 100%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could shorten the median time to effective antibiotic therapy by 20 h and 26 h, respectively, compared to standard AST. Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms and to predict AST results for K. pneumoniae isolates. Bioinformatics improvements enabling real-time alignment, coupled with rapid extraction and library preparation, will further enhance the accuracy and workflow of the Nanopore real-time approach.

evolution of the virus but also the fundamental mechanisms by which control measures affected its epidemic spread. These efforts complement the information provided by the rapidly growing public databases of SARS-CoV-2 sequences by focusing the collection of genomic data in settings where we can access extensive current and past clinical data to investigate fundamental questions about this evolving virus's changing relationship with human health. MethodsData availability. Raw nanopore and Illumina data are deposited at SRA (BioProject PRJNA629390). Consensus sequences are deposited at GISAID and Genbank (MT509452-MT509493, and MT646048-MT646120) under BioProject PRJNA650037 (accession numbers available in Supplemental Table 3).Specimens and patient data. Clinical specimens used for genetic characterization were remnant nasopharyngeal swabs available at the completion of standard of care testing at the Johns Hopkins Hospital clinical virology laboratory. In total, 143 samples were selected for analysis based on their distribution throughout March 2020 and representation of the range of disease severity observed during this period. During this period, automated patient metadata extraction was limited to the date a sample was confirmed positive; all other data required patient chart reviews. Samples were sequenced in 2 phases, with the first phase enriched for patients admitted to the ICU (14 of 55 samples collected March 11-21), and the second a convenience sample that captured as many samples as possible for sequencing, irrespective of disease severity or ICU admission (10 of 88 samples collected March 13 -31).Clinical data analysis. Data including patient demographics, symptoms, comorbidities, COVID-19 exposure, recent travel history, and results of chest imaging at presentation were abstracted from the electronic medical record (EMR). COVID-19 treatment (medication, supplemental oxygen, and invasive mechanical ventilation) and outcomes (home observation without inpatient admission, discharge after admission, ongoing admission, and death) were also abstracted from the EMR. Race as self-reported by the patient and documented in the EMR was collected in prespecified categories. Patients who reported (a) contact with an individual known to be COVID-19-infected or (b) high-risk exposure (e.g., healthcare worker) were classified as COVID-19-exposed. Comorbidities were assessed based on diagnoses in the EMR (i.e., diabetes, obesity, or alcohol use disorder) and further categorized for lung disease (e.g., asthma, COPD), cardiac disease (e.g., valvular heart disease, arrhythmias, hypertension), and immunocompromised (e.g., HIV positive, hematologic malignancy, solid organ transplant).Nucleic acid extraction. Automated nucleic acid extraction was performed using either the NucliSENS easy-Mag or eMAG instruments (bioMérieux) using software version 2.1.0.1. easyMag or eMAG lysis buffer (2 mL) was added to 500 μL of aliquoted viral transport media in a biosafety cabinet in either a BSL-3 or BSL-2 facility using BSL-3 biosafe...

The tobacco hornworm, Manduca sexta, is a lepidopteran insect that is used extensively as a model system for studying insect biology, development, neuroscience, and immunity. However, current studies rely on the highly fragmented reference genome Msex_1.0, which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies. We present a new reference genome for M. sexta, JHU_Msex_v1.0, applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly is 470 Mb and is ∼20× more continuous than the original assembly, with scaffold N50 &gt; 14 Mb. We annotated the assembly by lifting over existing annotations and supplementing with additional supporting RNA-based data for a total of 25,256 genes. The new reference assembly is accessible in annotated form for public use. We demonstrate that improved continuity of the M. sexta genome improves resequencing studies and benefits future research on M. sexta as a model organism.