Explore the vast range of titles available.

A human genome is sequenced and assembled de novo using a pocket-sized nanopore device. We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ∼30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ∼3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ∼6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.

This multiplex PCR enrichment protocol enables sequencing of Zika and other viral genomes of low abundance from clinical samples using the Illumina platform, or the portable MinION sequencer, facilitating direct application in field situations. Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1–2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab.

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 persist 1 , 2 . Here we present a human genome assembly that surpasses the continuity of GRCh38 2 , 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 chromosome 3 , 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. High-coverage, ultra-long-read nanopore sequencing is used to create a new human genome assembly that improves on the coverage and accuracy of the current reference (GRCh38) and includes the gap-free, telomere-to-telomere sequence of the X chromosome.

[...]an article from the New York Times highlights that actual student spending on course materials, including textbooks, was about half the actual cost of the textbooks and related course materials.6 Therefore, we divide the calculated savings by two and report that total as a more accurate representation of student savings. [...]we claim that students have saved about $11 million since IU's e-texts program started in spring 2012. According to page view records between the spring 2012 and spring 2016 semesters, 3,224 students from 251 courses (745 separate sections) printed over 130,000 pages of e-text (excluding multiple prints of the same page). [...]the sections below present data between the spring 2013 and spring 2016 semesters, broken down by distinct courses, sections, e-text titles, and students (table 2).

Since the advent of long read sequencing, achieving longer read lengths has been a key goal for many users. Ultra-long read sets (N50 ≥ 100 kb) produced from Oxford Nanopore sequencers have improved genome assemblies in recent years. However, despite progress in extraction protocols and library preparation methods, ultra-long sequencing remains challenging for many sample types. Here we compare various methods and introduce the FindingNemo protocol that: (1) optimises ultra-high molecular weight (UHMW) DNA extraction and library clean-up by using glass beads and Hexamminecobalt(III) chloride (CoHex), (2) can deliver high ultra-long sequencing yield of >20 Gb of reads from a single MinION flow cell or >100 Gb from PromethION devices (R9.4 to R10.4 pore variants), and (3) is scalable to using fewer input cells or lower DNA amounts, with extraction to sequencing possible in a single working day. By comparison, we show this protocol is superior to previous ones due to precise determination of input DNA quantity and quality by cell count, sample dilution and homogenization approaches.

The COVID-19 pandemic exposed vulnerabilities in global laboratory supply chains, disrupting genomic surveillance efforts essential to epidemic response. To address this challenge, we developed ARTIC HELP (Homebrew Enzymes for Library Preparation), a practical, open-source adaptation of the widely adopted ARTIC nanopore sequencing protocol for viral genomic surveillance. We describe generic, cost-effective alternatives to all enzyme mixes used in tiling multiplex RT-PCR amplification of the virus genome, and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL), making it broadly applicable to any laboratory. Through systematic evaluation, we identified a wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases as effective alternatives when standard reagents are unavailable due to high cost or limited supply: B-family Pfu-based polymerases with a fused Sso7d DNA-binding domain, and blends combining A-family (Taq-based) and B-family (Pfu-based) polymerases. Validation on clinical samples of SARS-CoV-2 and Norovirus GII confirmed that the HELP workflow achieves genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct ≤28), the wild-type M-MLV RT combined with selected Pfu or A+B polymerases, along with optimised HELP mixes (EP, BL, AL), achieved genome coverage of 84.0-99.6%. For Norovirus GII (Ct ≤32), the HELP workflow using one of the Pfu polymerases achieved genome coverage of >85% for six out of eight genotypes tested. Notably, several of the other polymerases tested showed reduced performance at higher Ct values. However, they still achieved strong coverage at Ct <24, supporting their use as emergency alternatives in rapid outbreak-response sequencing when viral input is high and RNA quality is sufficient. Our approach, ARTIC HELP, provides a framework which can be implemented to address supply chain disruptions, while maintaining robust genomic sequencing capabilities. A cost analysis highlights the well-known significant global disparities in reagent pricing, driven not by protocol differences but by import fees and supply barriers. Thus, our findings highlight the need for fairer global pricing models and support for local sourcing strategies like HELP, to promote equity in genomic research and ensure preparedness for future public health challenges.

[...]some students (30%) and faculty (20%) voiced concerns about using demographic data. Concerns of Bias and Equity in the Uses of Learner Data Across the system of stakeholders in higher education lies a spectrum of power in which students, who are the sources of the data, are frequently left vulnerable and unaware of how their behaviors, characteristics, and outcomes are used for research and evaluation purposes. [...]a second analysis looked to center the student voice by initiating an analysis with student responses (n = 20) to the following two questions: * To what degree are you concerned with issues of bias in the uses of learner data? * To what degree are you concerned with issues of equity in the uses of learner data? [...]in response to both questions, students referenced relationships with people at the institution, including faculty and staff who were specifically responsible for collecting and analyzing learning data. [...]a recurring ambiguity in student responses speaks to a possible gap between student conceptions of bias and equity in the uses of learner data and the practices institutions and researchers are using to engage with learning data in addressing potential areas of bias and equity. [...]acting on these data requires careful consideration of institutional responsibility in clarifying issues of bias and equity in learning data and empowering learners with the agency to engage meaningfully with these issues. The themes that arose from the analysis are summarized in six high-level barriers, as shown in table 1: * Availability of useful data (mentioned in 95% of responses) * Data literacy (50%) * Lack of process and strategy (50%) * Time and effort (32%) * Philosophical resistance or skepticism (32%) * Privacy, security, and misuse (32%) To generate potential strategies that could overcome these barriers, we presented our findings at the 2021 EDUCAUSE Learning Initiative Annual Meeting and solicited ideas from session participants via a collaborative document.2 (Because the document was anonymous, the ideas cannot be attributed.) The table also includes an excerpt of strategies generated at the session.

Background: The MinION is a new, portable single-molecule sequencer developed by Oxford Nanopore Technologies. It measures four inches in length and is powered from the USB 3.0 port of a laptop computer. By measuring the change in current produced when DNA strands translocate through and interact with a charged protein nanopore the device is able to deduce the underlying nucleotide sequence. Findings: We present a read dataset from whole-genome shotgun sequencing of the model organism Escherichia coli K-12 substr. MG1655 generated on a MinION device during the early-access MinION Access Program (MAP). Sequencing runs of the MinION are presented, one generated using R7 chemistry (released in July 2014) and one using R7.3 (released in September 2014). Conclusions: Base-called sequence data are provided to demonstrate the nature of data produced by the MinION platform and to encourage the development of customised methods for alignment, consensus and variant calling, de novo assembly and scaffolding. FAST5 files containing event data within the HDF5 container format are provided to assist with the development of improved base-calling methods. Datasets are provided through the GigaDB database at http://gigadb.org/dataset/100102

São Paulo (SP), a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in SP, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in SP, we generated and analysed virus genomic data and epizootic case data from NHP in SP. We report the occurrence of three spatiotemporally distinct phases of the outbreak in SP prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in SP, mostly sampled from non-human primates between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in SP state at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern SP subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of SP state. Our results shed light on the sylvatic transmission of yellow fever in highly fragmented forested regions in SP state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species. Competing Interest Statement The authors have declared no competing interest.

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38, along with the first 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 chromosome, we reconstructed the ~2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes. Footnotes * https://github.com/nanopore-wgs-consortium/CHM13 * http://www.stowers.org/research/publications/libpb-1453