Longitudinal Large-Scale Semiquantitative Proteomic Data Stability Across Multiple Instrument Platforms.

With the rapid developments in mass spectrometry (MS)-based proteomics methods, label-free semiquantitative proteomics has become an increasingly popular tool for profiling global protein abundances in an unbiased manner. However, the reproducibility of these data across time and LC-MS platforms is not well characterized. Here, we evaluate the performance of three LC-MS platforms (Orbitrap Elite, Q Exactive HF, and Orbitrap Fusion) in label-free semiquantitative analysis of cell surface proteins over a six-year period. Sucrose gradient ultracentrifugation was used for surfaceome enrichment, following gel separation for in-depth protein identification. With our established workflow, we consistently detected and reproducibly quantified >2300 putative cell surface proteins in a human acute myeloid leukemia (AML) cell line on all three platforms. To our knowledge this is the first study reporting highly reproducible semiquantitative proteomic data collection of biological replicates across multiple years and LC-MS platforms. These data provide experimental justification for semiquantitative proteomic study designs that are executed over multiyear time intervals and on different platforms. Multiyear and multiplatform experimental designs will likely enable larger scale proteomic studies and facilitate longitudinal proteomic studies by investigators lacking access to high throughput MS facilities. Data are available via ProteomeXchange with identifier PXD022721.

Mapping copy number variation by population-scale genome sequencing.

Genomic structural variants (SVs) are abundant in humans, differing from other forms of variation in extent, origin and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (that is, copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analysing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

Comparative and demographic analysis of orang-utan genomes.

'Orang-utan' is derived from a Malay term meaning 'man of the forest' and aptly describes the southeast Asian great apes native to Sumatra and Borneo. The orang-utan species, Pongo abelii (Sumatran) and Pongo pygmaeus (Bornean), are the most phylogenetically distant great apes from humans, thereby providing an informative perspective on hominid evolution. Here we present a Sumatran orang-utan draft genome assembly and short read sequence data from five Sumatran and five Bornean orang-utan genomes. Our analyses reveal that, compared to other primates, the orang-utan genome has many unique features. Structural evolution of the orang-utan genome has proceeded much more slowly than other great apes, evidenced by fewer rearrangements, less segmental duplication, a lower rate of gene family turnover and surprisingly quiescent Alu repeats, which have played a major role in restructuring other primate genomes. We also describe a primate polymorphic neocentromere, found in both Pongo species, emphasizing the gradual evolution of orang-utan genome structure. Orang-utans have extremely low energy usage for a eutherian mammal, far lower than their hominid relatives. Adding their genome to the repertoire of sequenced primates illuminates new signals of positive selection in several pathways including glycolipid metabolism. From the population perspective, both Pongo species are deeply diverse; however, Sumatran individuals possess greater diversity than their Bornean counterparts, and more species-specific variation. Our estimate of Bornean/Sumatran speciation time, 400,000 years ago, is more recent than most previous studies and underscores the complexity of the orang-utan speciation process. Despite a smaller modern census population size, the Sumatran effective population size (N(e)) expanded exponentially relative to the ancestral N(e) after the split, while Bornean N(e) declined over the same period. Overall, the resources and analyses presented here offer new opportunities in evolutionary genomics, insights into hominid biology, and an extensive database of variation for conservation efforts.

Genome analysis of the platypus reveals unique signatures of evolution.

We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome.

Acute myeloid leukaemia is a highly malignant haematopoietic tumour that affects about 13,000 adults in the United States each year. The treatment of this disease has changed little in the past two decades, because most of the genetic events that initiate the disease remain undiscovered. Whole-genome sequencing is now possible at a reasonable cost and timeframe to use this approach for the unbiased discovery of tumour-specific somatic mutations that alter the protein-coding genes. Here we present the results obtained from sequencing a typical acute myeloid leukaemia genome, and its matched normal counterpart obtained from the same patient's skin. We discovered ten genes with acquired mutations; two were previously described mutations that are thought to contribute to tumour progression, and eight were new mutations present in virtually all tumour cells at presentation and relapse, the function of which is not yet known. Our study establishes whole-genome sequencing as an unbiased method for discovering cancer-initiating mutations in previously unidentified genes that may respond to targeted therapies.

Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans.

Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1) gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.

The Open Pediatric Cancer Project.

Background: In 2019, the Open Pediatric Brain Tumor Atlas (OpenPBTA) was created as a global, collaborative open-science initiative to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Here, we extend the OpenPBTA to create the Open Pediatric Cancer (OpenPedCan) Project, a harmonized open-source multi-omic dataset from 6,112 pediatric cancer patients with 7,096 tumor events across more than 100 histologies. Combined with RNA-Seq from the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA), OpenPedCan contains nearly 48,000 total biospecimens (24,002 tumor and 23,893 normal specimens). Findings: We utilized Gabriella Miller Kids First (GMKF) workflows to harmonize WGS, WXS, RNA-seq, and Targeted Sequencing datasets to include somatic SNVs, InDels, CNVs, SVs, RNA expression, fusions, and splice variants. We integrated summarized CPTAC whole cell proteomics and phospho-proteomics data, miRNA-Seq data, and have developed a methylation array harmonization workflow to include m-values, beta-vales, and copy number calls. OpenPedCan contains reproducible, dockerized workflows in GitHub, CAVATICA, and Amazon Web Services (AWS) to deliver harmonized and processed data from over 60 scalable modules which can be leveraged both locally and on AWS. The processed data are released in a versioned manner and accessible through CAVATICA or AWS S3 download (from GitHub), and queryable through PedcBioPortal and the NCI's pediatric Molecular Targets Platform. Notably, we have expanded PBTA molecular subtyping to include methylation information to align with the WHO 2021 Central Nervous System Tumor classifications, allowing us to create research- grade integrated diagnoses for these tumors. Conclusions: OpenPedCan data and its reproducible analysis module framework are openly available and can be utilized and/or adapted by researchers to accelerate discovery, validation, and clinical translation.

Evolution of genes and genomes on the Drosophila phylogeny.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Using human factors principles to redesign a 3D lab workflow during the COVID-19 pandemic.

BACKGROUND: Like most hospitals, our hospital experienced COVID-19 pandemic-related supply chain shortages. Our additive manufacturing lab's capacity to offset these shortages was soon overwhelmed, leading to a need to improve the efficiency of our existing workflow. We undertook a work system analysis guided by the Systems Engineering Initiative for Patient Safety (SEIPS) construct which is based on human factors and quality improvement principles. Our objective was to understand the inefficiencies in project submission, review, and acceptance decisions, and make systematic improvements to optimize lab operations. METHODS: Contextual inquiry (interviews and workflow analysis) revealed suboptimal characteristics of the system, specifically, reliance on a single person to facilitate work and, at times, fractured communication with project sponsors, with root causes related to the project intake and evaluation process as identified through SEIPS tools. As interventions, the analysis led us to: 1) enhance an existing but underused project submission form, 2) design and implement an internal project scorecard to standardize evaluation of requests, and 3) distribute the responsibility of submission evaluation across lab members. We implemented these interventions in May 2021 for new projects and compare them to our baseline February 1, 2018 through - April 30, 2021 performance (1184 days). RESULTS: All project requests were submitted using the enhanced project submission form and all received a standardized evaluation with the project scorecard. Prior to interventions, we completed 35/79 (44%) of projects, compared to 12/20 (60%) of projects after interventions were implemented. Time to review new submissions was reduced from an average of 58 days to 4 days. A more distributed team responsibility structure permitted improved workflow with no increase in staffing, allowing the Lab Manager to devote more time to engineering rather than administrative/decision tasks. CONCLUSIONS: By optimizing our workflows utilizing a human factors approach, we improved the work system of our additive manufacturing lab to be responsive to the urgent needs of the pandemic. The current workflow provides insights for labs aiming to meet the growing demand for point-of-care manufacturing.

Additive manufacturing (3d printing) in response to a pandemic: Lessons learned at the children's hospital of Philadelphia.

The COVID-19 pandemic produced unprecedented challenges to healthcare and medical device manufacturing (e.g. personal protective device and replacement part shortages). Additive manufacturing, 3D printing, and the maker community were uniquely positioned to respond to these needs by providing in-house design and manufacturing to meet the needs of clinicians and hospitals. This paper reviews the pandemic response of Children's Hospital of Philadelphia CHAMP 3D Lab, a point-ofcare3D printing team that supports clinical and research projects across the hospital network. The CHAMP team responded to a variety of COVID-19 healthcare needs including providing protective eyewear and ventilator components, creating a transport hook, and designing a novel transparent facemask. This case series details our response to these needs, describing challenges experienced and lessons learned in overcoming them so that others may learn from our experiences. Challenges to responding to the pandemic included the need to handle urgent pandemic related requests in addition to our standard fare. This required us to not only expand our capacity without additional resources, but also to develop a system of prioritization. Specific changes made included: streamlining workflows, identifying safety review processes, and developing/enlisting a network of collaborators. Further, we consider how to transition to a future, post-pandemic world without losing the cohesive drive of emergency-induced innovation. This paper aims to share what we have learned and to encourage both teams currently engaged in the printing community and those looking to join it.

Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny.

To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism-based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80-110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently.

A physical map of the chicken genome.

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones. In an accompanying paper we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes.

Application of a superword array in genome assembly.

We introduce a data structure called a superword array for finding quickly matches between DNA sequences. The superword array possesses some desirable features of the lookup table and suffix array. We describe simple algorithms for constructing and using a superword array to find pairs of sequences that share a unique superword. The algorithms are implemented in a genome assembly program called PCAP.REP for computation of overlaps between reads. Experimental results produced by PCAP.REP and PCAP on a whole-genome dataset show that PCAP.REP produced a more accurate and contiguous assembly than PCAP.

Design and implementation of a generalized laboratory data model.

BACKGROUND: Investigators in the biological sciences continue to exploit laboratory automation methods and have dramatically increased the rates at which they can generate data. In many environments, the methods themselves also evolve in a rapid and fluid manner. These observations point to the importance of robust information management systems in the modern laboratory. Designing and implementing such systems is non-trivial and it appears that in many cases a database project ultimately proves unserviceable. RESULTS: We describe a general modeling framework for laboratory data and its implementation as an information management system. The model utilizes several abstraction techniques, focusing especially on the concepts of inheritance and meta-data. Traditional approaches commingle event-oriented data with regular entity data in ad hoc ways. Instead, we define distinct regular entity and event schemas, but fully integrate these via a standardized interface. The design allows straightforward definition of a "processing pipeline" as a sequence of events, obviating the need for separate workflow management systems. A layer above the event-oriented schema integrates events into a workflow by defining "processing directives", which act as automated project managers of items in the system. Directives can be added or modified in an almost trivial fashion, i.e., without the need for schema modification or re-certification of applications. Association between regular entities and events is managed via simple "many-to-many" relationships. We describe the programming interface, as well as techniques for handling input/output, process control, and state transitions. CONCLUSION: The implementation described here has served as the Washington University Genome Sequencing Center's primary information system for several years. It handles all transactions underlying a throughput rate of about 9 million sequencing reactions of various kinds per month and has handily weathered a number of major pipeline reconfigurations. The basic data model can be readily adapted to other high-volume processing environments.

The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics.

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.

Nematode.net: a tool for navigating sequences from parasitic and free-living nematodes.

Nematode.net (www.nematode.net) is a web- accessible resource for investigating gene sequences from nematode genomes. The database is an outgrowth of the parasitic nematode EST project at Washington University's Genome Sequencing Center (GSC), St Louis. A sister project at the University of Edinburgh and the Sanger Institute is also underway. More than 295,000 ESTs have been generated from >30 nematodes other than Caenorhabditis elegans including key parasites of humans, animals and plants. Nematode.net currently provides NemaGene EST cluster consensus sequence, enhanced online BLAST search tools, functional classifications of cluster sequences and comprehensive information concerning the ongoing generation of nematode genome data. The long-term goal of nematode.net is to provide the scientific community with the highest quality sequence information and tools for studying these diverse species.

A catalog of reference genomes from the human microbiome.

The human microbiome refers to the community of microorganisms, including prokaryotes, viruses, and microbial eukaryotes, that populate the human body. The National Institutes of Health launched an initiative that focuses on describing the diversity of microbial species that are associated with health and disease. The first phase of this initiative includes the sequencing of hundreds of microbial reference genomes, coupled to metagenomic sequencing from multiple body sites. Here we present results from an initial reference genome sequencing of 178 microbial genomes. From 547,968 predicted polypeptides that correspond to the gene complement of these strains, previously unidentified ("novel") polypeptides that had both unmasked sequence length greater than 100 amino acids and no BLASTP match to any nonreference entry in the nonredundant subset were defined. This analysis resulted in a set of 30,867 polypeptides, of which 29,987 (approximately 97%) were unique. In addition, this set of microbial genomes allows for approximately 40% of random sequences from the microbiome of the gastrointestinal tract to be associated with organisms based on the match criteria used. Insights into pan-genome analysis suggest that we are still far from saturating microbial species genetic data sets. In addition, the associated metrics and standards used by our group for quality assurance are presented.

The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism.

Here we present a draft genome sequence of the nematode Pristionchus pacificus, a species that is associated with beetles and is used as a model system in evolutionary biology. With 169 Mb and 23,500 predicted protein-coding genes, the P. pacificus genome is larger than those of Caenorhabditis elegans and the human parasite Brugia malayi. Compared to C. elegans, the P. pacificus genome has more genes encoding cytochrome P450 enzymes, glucosyltransferases, sulfotransferases and ABC transporters, many of which were experimentally validated. The P. pacificus genome contains genes encoding cellulase and diapausin, and cellulase activity is found in P. pacificus secretions, indicating that cellulases can be found in nematodes beyond plant parasites. The relatively higher number of detoxification and degradation enzymes in P. pacificus is consistent with its necromenic lifestyle and might represent a preadaptation for parasitism. Thus, comparative genomics analysis of three ecologically distinct nematodes offers a unique opportunity to investigate the association between genome structure and lifestyle.

Physical map-assisted whole-genome shotgun sequence assemblies.

We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the layout phase of WGS assemblies. This process is facilitated by FASSI, a stand-alone application that calculates BAC end and BAC overlap length constraints from clone fingerprint map contigs created by the FPC package. FASSI is designed to work with the assembly tool PCAP, but its output can be formatted to work with other WGS assembly algorithms able to use length constraints for individual clones. The FASSI method is simple to implement, potentially cost-effective, and has resulted in the increase of scaffold contiguity for both the Drosophila melanogaster and Cryptococcus gattii genomes when compared to a control assembly without map-derived constraints. A 6.5-fold coverage draft DNA sequence of the Pan troglodytes (chimpanzee) genome was assembled using map-derived constraints and resulted in a 26.1% increase in scaffold contiguity.