Implementation of California COVIDNet - a multi-sector collaboration for statewide SARS-CoV-2 genomic surveillance.

Introduction: The SARS-CoV-2 pandemic represented a formidable scientific and technological challenge to public health due to its rapid spread and evolution. To meet these challenges and to characterize the virus over time, the State of California established the California SARS-CoV-2 Whole Genome Sequencing (WGS) Initiative, or "California COVIDNet". This initiative constituted an unprecedented multi-sector collaborative effort to achieve large-scale genomic surveillance of SARS-CoV-2 across California to monitor the spread of variants within the state, to detect new and emerging variants, and to characterize outbreaks in congregate, workplace, and other settings. Methods: California COVIDNet consists of 50 laboratory partners that include public health laboratories, private clinical diagnostic laboratories, and academic sequencing facilities as well as expert advisors, scientists, consultants, and contractors. Data management, sample sourcing and processing, and computational infrastructure were major challenges that had to be resolved in the midst of the pandemic chaos in order to conduct SARS-CoV-2 genomic surveillance. Data management, storage, and analytics needs were addressed with both conventional database applications and newer cloud-based data solutions, which also fulfilled computational requirements. Results: Representative and randomly selected samples were sourced from state-sponsored community testing sites. Since March of 2021, California COVIDNet partners have contributed more than 450,000 SARS-CoV-2 genomes sequenced from remnant samples from both molecular and antigen tests. Combined with genomes from CDC-contracted WGS labs, there are currently nearly 800,000 genomes from all 61 local health jurisdictions (LHJs) in California in the COVIDNet sequence database. More than 5% of all reported positive tests in the state have been sequenced, with similar rates of sequencing across 5 major geographic regions in the state. Discussion: Implementation of California COVIDNet revealed challenges and limitations in the public health system. These were overcome by engaging in novel partnerships that established a successful genomic surveillance program which provided valuable data to inform the COVID-19 public health response in California. Significantly, California COVIDNet has provided a foundational data framework and computational infrastructure needed to respond to future public health crises.

Germline variants of ATG7 in familial cholangiocarcinoma alter autophagy and p62.

Autophagy is a housekeeping mechanism tasked with eliminating misfolded proteins and damaged organelles to maintain cellular homeostasis. Autophagy deficiency results in increased oxidative stress, DNA damage and chronic cellular injury. Among the core genes in the autophagy machinery, ATG7 is required for autophagy initiation and autophagosome formation. Based on the analysis of an extended pedigree of familial cholangiocarcinoma, we determined that all affected family members had a novel germline mutation (c.2000C>T p.Arg659* (p.R659*)) in ATG7. Somatic deletions of ATG7 were identified in the tumors of affected individuals. We applied linked-read sequencing to one tumor sample and demonstrated that the ATG7 somatic deletion and germline mutation were located on distinct alleles, resulting in two hits to ATG7. From a parallel population genetic study, we identified a germline polymorphism of ATG7 (c.1591C>G p.Asp522Glu (p.D522E)) associated with increased risk of cholangiocarcinoma. To characterize the impact of these germline ATG7 variants on autophagy activity, we developed an ATG7-null cell line derived from the human bile duct. The mutant p.R659* ATG7 protein lacked the ability to lipidate its LC3 substrate, leading to complete loss of autophagy and increased p62 levels. Our findings indicate that germline ATG7 variants have the potential to impact autophagy function with implications for cholangiocarcinoma development.

Closed Genome Sequence of Clostridium botulinum Strain IBCA10-7060 Type Bh.

Clostridium botulinum strain IBCA10-7060 was isolated from a stool specimen from an infant botulism patient and is the only Clostridium botulinum strain known that produces botulinum toxin type H. We present here its 4.09-Mbp closed genome sequence.

Unique k-mer sequences for validating cancer-related substitution, insertion and deletion mutations.

Cancer genome sequencing has led to important discoveries such as the identification of cancer genes. However, challenges remain in the analysis of cancer genome sequencing. One significant issue is that mutations identified by multiple variant callers are frequently discordant even when using the same genome sequencing data. For insertion and deletion mutations, oftentimes there is no agreement among different callers. Identifying somatic mutations involves read mapping and variant calling, a complicated process that uses many parameters and model tuning. To validate the identification of true mutations, we developed a method using k-mer sequences. First, we characterized the landscape of unique versus non-unique k-mers in the human genome. Second, we developed a software package, KmerVC, to validate the given somatic mutations from sequencing data. Our program validates the occurrence of a mutation based on statistically significant difference in frequency of k-mers with and without a mutation from matched normal and tumor sequences. Third, we tested our method on both simulated and cancer genome sequencing data. Counting k-mer involving mutations effectively validated true positive mutations including insertions and deletions across different individual samples in a reproducible manner. Thus, we demonstrated a straightforward approach for rapidly validating mutations from cancer genome sequencing data.

High density mechanical energy storage with carbon nanothread bundle.

The excellent mechanical properties of carbon nanofibers bring promise for energy-related applications. Through in silico studies and continuum elasticity theory, here we show that the ultra-thin carbon nanothreads-based bundles exhibit a high mechanical energy storage density. Specifically, the gravimetric energy density is found to decrease with the number of filaments, with torsion and tension as the two dominant contributors. Due to the coupled stresses, the nanothread bundle experiences fracture before reaching the elastic limit of any individual deformation mode. Our results show that nanothread bundles have similar mechanical energy storage capacity compared to (10,10) carbon nanotube bundles, but possess their own advantages. For instance, the structure of the nanothread allows us to realize the full mechanical energy storage potential of its bundle structure through pure tension, with a gravimetric energy density of up to 1.76 MJ kg-1, which makes them appealing alternative building blocks for energy storage devices.

Whole genome analysis identifies the association of TP53 genomic deletions with lower survival in Stage III colorectal cancer.

DNA copy number aberrations (CNA) are frequently observed in colorectal cancers (CRC). There is an urgent need for CNA-based biomarkers in clinics,. n For Stage III CRC, if combined with imaging or pathologic evidence, these markers promise more precise care. We conducted this Stage III specific biomarker discovery with a cohort of 134 CRCs, and with a newly developed high-efficiency CNA profiling protocol. Specifically, we developed the profiling protocol for tumor-normal matched tissue samples based on low-coverage clinical whole-genome sequencing (WGS). We demonstrated the protocol's accuracy and robustness by a systematic benchmark with microarray, high-coverage whole-exome and -genome approaches, where the low-coverage WGS-derived CNA segments were highly accordant (PCC >0.95) with those derived from microarray, and they were substantially less variable if compared to exome-derived segments. A lasso-based model and multivariate cox regression analysis identified a chromosome 17p loss, containing the TP53 tumor suppressor gene, that was significantly associated with reduced survival (P = 0.0139, HR = 1.688, 95% CI = [1.112-2.562]), which was validated by an independent cohort of 187 Stage III CRCs. In summary, this low-coverage WGS protocol has high sensitivity, high resolution and low cost and the identified 17p-loss is an effective poor prognosis marker for Stage III patients.

Haplotype-resolved and integrated genome analysis of the cancer cell line HepG2.

HepG2 is one of the most widely used human cancer cell lines in biomedical research and one of the main cell lines of ENCODE. Although the functional genomic and epigenomic characteristics of HepG2 are extensively studied, its genome sequence has never been comprehensively analyzed and higher order genomic structural features are largely unknown. The high degree of aneuploidy in HepG2 renders traditional genome variant analysis methods challenging and partially ineffective. Correct and complete interpretation of the extensive functional genomics data from HepG2 requires an understanding of the cell line's genome sequence and genome structure. Using a variety of sequencing and analysis methods, we identified a wide spectrum of genome characteristics in HepG2: copy numbers of chromosomal segments at high resolution, SNVs and Indels (corrected for aneuploidy), regions with loss of heterozygosity, phased haplotypes extending to entire chromosome arms, retrotransposon insertions and structural variants (SVs) including complex and somatic genomic rearrangements. A large number of SVs were phased, sequence assembled and experimentally validated. We re-analyzed published HepG2 datasets for allele-specific expression and DNA methylation and assembled an allele-specific CRISPR/Cas9 targeting map. We demonstrate how deeper insights into genomic regulatory complexity are gained by adopting a genome-integrated framework.

Comprehensive, integrated, and phased whole-genome analysis of the primary ENCODE cell line K562.

K562 is widely used in biomedical research. It is one of three tier-one cell lines of ENCODE and also most commonly used for large-scale CRISPR/Cas9 screens. Although its functional genomic and epigenomic characteristics have been extensively studied, its genome sequence and genomic structural features have never been comprehensively analyzed. Such information is essential for the correct interpretation and understanding of the vast troves of existing functional genomics and epigenomics data for K562. We performed and integrated deep-coverage whole-genome (short-insert), mate-pair, and linked-read sequencing as well as karyotyping and array CGH analysis to identify a wide spectrum of genome characteristics in K562: copy numbers (CN) of aneuploid chromosome segments at high-resolution, SNVs and indels (both corrected for CN in aneuploid regions), loss of heterozygosity, megabase-scale phased haplotypes often spanning entire chromosome arms, structural variants (SVs), including small and large-scale complex SVs and nonreference retrotransposon insertions. Many SVs were phased, assembled, and experimentally validated. We identified multiple allele-specific deletions and duplications within the tumor suppressor gene FHIT Taking aneuploidy into account, we reanalyzed K562 RNA-seq and whole-genome bisulfite sequencing data for allele-specific expression and allele-specific DNA methylation. We also show examples of how deeper insights into regulatory complexity are gained by integrating genomic variant information and structural context with functional genomics and epigenomics data. Furthermore, using K562 haplotype information, we produced an allele-specific CRISPR targeting map. This comprehensive whole-genome analysis serves as a resource for future studies that utilize K562 as well as a framework for the analysis of other cancer genomes.

Tuning the Amount of Oxygen Vacancies in Sputter-Deposited SnOx films for Enhancing the Performance of Perovskite Solar Cells.

This work demonstrates the effect of oxygen vacancies in SnOx thin films on the performance of perovskite solar cells. Various SnOx films with different amounts of oxygen vacancies were deposited by sputtering at different substrate temperatures (25-300 °C). The transmittance of the films decreased from 82 to 66 % with increasing deposition temperature from 25 to 300 °C. Both X-ray photoelectron spectroscopy and electron-spin resonance spectroscopy confirmed that a higher density of oxygen vacancies was created within the SnOx film at a high substrate temperature, which caused narrowing of the SnOx bandgap from 4.1 (25 °C) to 3.74 eV (250 °C). Combined ultraviolet photoelectron spectroscopy and UV/Vis spectroscopy showed an excellent conduction band position alignment between the methylammonium lead iodide perovskite layer (3.90 eV) and the SnOx electron transport layer deposited at 250 °C (3.92 eV). As a result, a significant enhancement of the open-circuit voltage from 0.82 to 1.0 V was achieved, resulting in an increase of the power conversion efficiency of the perovskite solar cells from 11 to 14 %. This research demonstrated a facile approach for controlling the amount of oxygen vacancies in SnOx thin films to achieve a desirable energy alignment with the perovskite absorber layer for enhanced device performance.

Vinylene and benzo[c][1,2,5]thiadiazole: effect of the π-spacer unit on the properties of bis(2-oxoindolin-3-ylidene)-benzodifuran-dione containing polymers for n-channel organic field-effect transistors.

Two polymers based on (3E,7E)-3,7-bis(2-oxoindolin-3-ylidene)benzo[1,2-b:4,5-b']difuran-2,6(3H,7H)-dione (BIBDF) coupled with (E)-2-(2-(thiophen-2-yl)vinyl)thiophene (TVT) or dithienylbenzothiadiazole (TBT), namely PBIBDF-TVT and PBIBDF-TBT were synthesized via the Stille coupling reaction. The effect of benzothiadiazole or vinylene-π spacer of the copolymers on optical properties, energy levels, electronic device performance and microstructure were studied. It was found that PBIBDF-TBT based OFET devices, annealed at 180 °C, showed better performance with the highest electron mobility of 2.9 × 10-2 cm2 V s-1 whereas PBIBDF-TVT polymer exhibited 5.0 × 10-4 cm2 V s-1. The two orders of magnitude higher electron mobility of PBIBDF-TBT over PBIBDT-TVT is a clear indicator of the better charge transport ability of this polymer semiconductor arising from its higher crystallinity and better donor-acceptor interaction.

Identification of large rearrangements in cancer genomes with barcode linked reads.

Large genomic rearrangements involve inversions, deletions and other structural changes that span Megabase segments of the human genome. This category of genetic aberration is the cause of many hereditary genetic disorders and contributes to pathogenesis of diseases like cancer. We developed a new algorithm called ZoomX for analysing barcode-linked sequence reads-these sequences can be traced to individual high molecular weight DNA molecules (>50 kb). To generate barcode linked sequence reads, we employ a library preparation technology (10X Genomics) that uses droplets to partition and barcode DNA molecules. Using linked read data from whole genome sequencing, we identify large genomic rearrangements, typically greater than 200kb, even when they are only present in low allelic fractions. Our algorithm uses a Poisson scan statistic to identify genomic rearrangement junctions, determine counts of junction-spanning molecules and calculate a Fisher's exact test for determining statistical significance for somatic aberrations. Utilizing a well-characterized human genome, we benchmarked this approach to accurately identify large rearrangement. Subsequently, we demonstrated that our algorithm identifies somatic rearrangements when present in lower allelic fractions as occurs in tumors. We characterized a set of complex cancer rearrangements with multiple classes of structural aberrations and with possible roles in oncogenesis.

Chromosome-scale mega-haplotypes enable digital karyotyping of cancer aneuploidy.

Genomic instability is a frequently occurring feature of cancer that involves large-scale structural alterations. These somatic changes in chromosome structure include duplication of entire chromosome arms and aneuploidy where chromosomes are duplicated beyond normal diploid content. However, the accurate determination of aneuploidy events in cancer genomes is a challenge. Recent advances in sequencing technology allow the characterization of haplotypes that extend megabases along the human genome using high molecular weight (HMW) DNA. For this study, we employed a library preparation method in which sequence reads have barcodes linked to single HMW DNA molecules. Barcode-linked reads are used to generate extended haplotypes on the order of megabases. We developed a method that leverages haplotypes to identify chromosomal segmental alterations in cancer and uses this information to join haplotypes together, thus extending the range of phased variants. With this approach, we identified mega-haplotypes that encompass entire chromosome arms. We characterized the chromosomal arm changes and aneuploidy events in a manner that offers similar information as a traditional karyotype but with the benefit of DNA sequence resolution. We applied this approach to characterize aneuploidy and chromosomal alterations from a series of primary colorectal cancers.

Molecular Engineering Strategy for High Efficiency Fullerene-Free Organic Solar Cells Using Conjugated 1,8-Naphthalimide and Fluorenone Building Blocks.

We designed, synthesized, and characterized a series of novel electron deficient small molecule nonfullerene acceptors based on 1,8-naphthalimide (NAI) and 9-fluorenone (FN) with different branched alkyl chains using various techniques. These molecules are based on an acceptor-donor-acceptor-donor-acceptor (A1-D-A2-D-A1) molecular design configuration with NAI as the end-capping acceptor (A1), FN as electron-withdrawing central (A2) group, and thiophene ring as a donor (D) unit. These materials are named as NAI-FN-NAI (BO) and NAI-FN-NAI (HD) where BO and HD represent butyloctyl and hexyldecyl alkyl groups, respectively. To further modify energy levels of these materials, we converted the weak electron withdrawing ketonic group (C═O) attached to the FN moiety of NAI-FN-NAI (BO) to a stronger electron withdrawing cyano group (C≡N) to obtain the compound NAI-FCN-NAI (BO) by keeping the same alkyl chain. The optical, electrochemical, and thermal properties of the new acceptors were studied. The materials exhibited higher to medium band gaps, low lowest unoccupied molecular orbital (LUMO) energy levels, and highly thermally stable properties. Organic solar cell devices employing conventional poly(3-hexylthiophene) (P3HT) a donor polymer and the newly designed small molecules as the acceptor were investigated. Among all new materials, organic solar cell devices based on NAI-FN-NAI (BO) as an acceptor exhibit the highest performance with an open circuit voltage (VOC) of 0.88 V, a short-circuit current density (JSC) of 9.1 mAcm-2, a fill factor (FF) of 45%, and an overall power conversion efficiency (PCE) of 3.6%. This is the first report of 9-fluorenone based nonfullerene acceptor with P3HT donor in organic solar cell devices with such a promising performance.

A genome-wide approach for detecting novel insertion-deletion variants of mid-range size.

We present SWAN, a statistical framework for robust detection of genomic structural variants in next-generation sequencing data and an analysis of mid-range size insertion and deletions (<10 Kb) for whole genome analysis and DNA mixtures. To identify these mid-range size events, SWAN collectively uses information from read-pair, read-depth and one end mapped reads through statistical likelihoods based on Poisson field models. SWAN also uses soft-clip/split read remapping to supplement the likelihood analysis and determine variant boundaries. The accuracy of SWAN is demonstrated by in silico spike-ins and by identification of known variants in the NA12878 genome. We used SWAN to identify a series of novel set of mid-range insertion/deletion detection that were confirmed by targeted deep re-sequencing. An R package implementation of SWAN is open source and freely available.

From brittle to ductile: a structure dependent ductility of diamond nanothread.

As a potential building block for the next generation of devices/multifunctional materials that are spreading in almost every technology sector, one-dimensional (1D) carbon nanomaterial has received intensive research interests. Recently, a new ultra-thin diamond nanothread (DNT) has joined this palette, which is a 1D structure with poly-benzene sections connected by Stone-Wales (SW) transformation defects. Using large-scale molecular dynamics simulations, we found that this sp(3) bonded DNT can transition from brittle to ductile behaviour by varying the length of the poly-benzene sections, suggesting that DNT possesses entirely different mechanical responses than other 1D carbon allotropes. Analogously, the SW defects behave like a grain boundary that interrupts the consistency of the poly-benzene sections. For a DNT with a fixed length, the yield strength fluctuates in the vicinity of a certain value and is independent of the "grain size". On the other hand, both yield strength and yield strain show a clear dependence on the total length of DNT, which is due to the fact that the failure of the DNT is dominated by the SW defects. Its highly tunable ductility together with its ultra-light density and high Young's modulus makes diamond nanothread ideal for the creation of extremely strong three-dimensional nano-architectures.

Haplotyping germline and cancer genomes with high-throughput linked-read sequencing.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

Investigation on Temperature-Dependent Electrical Conductivity of Carbon Nanotube/Epoxy Composites for Sustainable Energy Applications.

Composites with carbon nanotubes are becoming increasingly used in energy storage and electronic devices, due to incorporated excellent properties from carbon nanotubes and polymers. Although their properties make them more attractive than conventional smart materials, their electrical properties have been found to be temperature-dependent which is important to consider for the design of devices. To study the effects of temperature in electrically conductive multi-wall carbon nanotube/epoxy composites, thin films were prepared and the effect of temperature on the resistivity, thermal properties and Raman spectral characteristics of the composite films was evaluated. Resistivity-temperature profiles showed three distinct regions in as-cured samples and only two regions in samples whose thermal histories had been erased. In the vicinity of the glass transition temperature, the as-cured composites exhibited pronounced resistivity and enthalpic relaxation peaks, which both disappeared after erasing the composites' thermal histories by temperature cycling. Combined DSC, Raman spectroscopy, and resistivity-temperature analyses indicated that this phenomenon can be attributed to the physical aging of the epoxy matrix and that, in the region of the observed thermal history-dependent resistivity peaks, structural rearrangement of the conductive carbon nanotube network occurs through a volume expansion/relaxation process. These results have led to an overall greater understanding of the temperature-dependent behaviour of conductive carbon nanotube/epoxy composites, including the positive temperature coefficient effect.

Emergence of Hemagglutinin Mutations During the Course of Influenza Infection.

Influenza remains a significant cause of disease mortality. The ongoing threat of influenza infection is partly attributable to the emergence of new mutations in the influenza genome. Among the influenza viral gene products, the hemagglutinin (HA) glycoprotein plays a critical role in influenza pathogenesis, is the target for vaccines and accumulates new mutations that may alter the efficacy of immunization. To study the emergence of HA mutations during the course of infection, we employed a deep-targeted sequencing method. We used samples from 17 patients with active H1N1 or H3N2 influenza infections. These patients were not treated with antivirals. In addition, we had samples from five patients who were analyzed longitudinally. Thus, we determined the quantitative changes in the fractional representation of HA mutations during the course of infection. Across individuals in the study, a series of novel HA mutations directly altered the HA coding sequence were identified. Serial viral sampling revealed HA mutations that either were stable, expanded or were reduced in representation during the course of the infection. Overall, we demonstrated the emergence of unique mutations specific to an infected individual and temporal genetic variation during infection.

Tuning the resonance properties of 2D carbon nanotube networks towards a mechanical resonator.

The capabilities of the mechanical resonator-based nanosensors in detecting ultra-small mass or force shifts have driven a continuing exploration of the palette of nanomaterials for such application purposes. Based on large-scale molecular dynamics simulations, we have assessed the applicability of a new class of carbon nanomaterials for nanoresonator usage, i.e. the single-wall carbon nanotube (SWNT) network. It is found that SWNT networks inherit excellent mechanical properties from the constituent SWNTs, possessing a high natural frequency. However, although a high quality factor is suggested from the simulation results, it is hard to obtain an unambiguous Q-factor due to the existence of vibration modes in addition to the dominant mode. The nonlinearities resulting from these extra vibration modes are found to exist uniformly under various testing conditions including different initial actuations and temperatures. Further testing shows that these modes can be effectively suppressed through the introduction of axial strain, leading to an extremely high quality factor in the order of 10(9) estimated from the SWNT network with 2% tensile strain. Additional studies indicate that the carbon rings connecting the SWNTs can also be used to alter the vibrational properties of the resulting network. This study suggests that the SWNT network can be a good candidate for applications as nanoresonators.

Allele-specific copy number profiling by next-generation DNA sequencing.

The progression and clonal development of tumors often involve amplifications and deletions of genomic DNA. Estimation of allele-specific copy number, which quantifies the number of copies of each allele at each variant loci rather than the total number of chromosome copies, is an important step in the characterization of tumor genomes and the inference of their clonal history. We describe a new method, falcon, for finding somatic allele-specific copy number changes by next generation sequencing of tumors with matched normals. falcon is based on a change-point model on a bivariate mixed Binomial process, which explicitly models the copy numbers of the two chromosome haplotypes and corrects for local allele-specific coverage biases. By using the Binomial distribution rather than a normal approximation, falcon more effectively pools evidence from sites with low coverage. A modified Bayesian information criterion is used to guide model selection for determining the number of copy number events. Falcon is evaluated on in silico spike-in data and applied to the analysis of a pre-malignant colon tumor sample and late-stage colorectal adenocarcinoma from the same individual. The allele-specific copy number estimates obtained by falcon allows us to draw detailed conclusions regarding the clonal history of the individual's colon cancer.