Biology subjects

Goodwin, S.

Publications and source records attributed to Goodwin, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Engineered T cells controlled by granulocyte colony-stimulating factor eradicate murine melanoma without lymphodepletion or interleukin-2

Tumour-infiltrating lymphocyte (TIL) therapy can be effective against metastatic melanoma, including immune checkpoint-resistant disease, but current protocols require lymphodepletion and high-dose interleukin-2 (IL-2), which can cause substantial toxicity. To overcome this limitation, we engineered a chimeric receptor, G2R, that generates an IL-2-like proliferative signal in response to granulocyte colony-stimulating factor (G-CSF), a well-tolerated cytokine widely used in oncology. Human T cells expressing G2R expanded in vitro in response to G-CSF in place of IL-2 or interleukin-15 (IL-15), and the three cytokines induced broadly similar intracellular signalling and cytokine secretion profiles. In immune-competent mice bearing established B16-F10 melanoma, adoptive transfer of G2R-expressing, tumour-reactive CD8+ T cells followed by systemic G-CSF treatment induced profound T cell expansion. This was accompanied by complete, durable tumour regression in over 75% of animals without the need for lymphodepletion, exogenous IL-2 or any other therapy. G-CSF treatment induced transient neutrophil expansion in peripheral blood but no increase in intratumoural neutrophils or macrophages. G2R-expressing memory T cells persisted long-term and could be re-expanded upon G-CSF re-treatment, enabling rejection of second tumour challenges. Thus, G-CSF can be repurposed to enable clinician-controlled anti-tumour T cell responses of profound magnitude, efficacy and durability, an approach that is readily translatable to humans.

immunology↗

bioRxiv2025

The role of MICOS in modulating mitochondrial dynamics and structural changes in vulnerable regions of Alzheimer's Disease

Mitochondrial contact site and cristae organizing system (MICOS) complexes are critical for maintaining the mitochondrial architecture, cristae integrity, and organelle communication in neurons. MICOS disruption has been implicated in neurodegenerative disorders, including Alzheimers disease (AD), yet the spatiotemporal dynamics of MICOS-associated neuronal alterations during aging remain unclear. Using three-dimensional reconstructions of hypothalamic and cortical neurons, we observed age-dependent fragmentation of mitochondrial cristae, reduced intermitochondrial connectivity, and compartment-specific changes in mitochondrial size and morphology. Notably, these structural deficits were most pronounced in neurons vulnerable to AD-related pathology, suggesting a mechanistic link between MICOS disruption and the early mitochondrial dysfunction observed in patients with AD. Our findings indicate that the loss of MICOS integrity is a progressive feature of neuronal aging, contributing to impaired bioenergetics and reduced resilience to metabolic stress and potentially facilitating neurodegenerative processes. MICOS disruption reduced neuronal firing and synaptic responsiveness, with miclxin treatment decreasing mitochondrial connectivity and inducing cristae disorganization. These changes link MICOS structural deficits directly to impaired neuronal excitability, highlighting vulnerability to AD-related neurodegeneration. These results underscore the importance of MICOS as a critical determinant of neuronal mitochondrial health and as a potential target for interventions aimed at mitigating AD-related mitochondrial dysfunction.

neuroscience↗

bioRxiv2025

Multi-site Assessment of Methods for Cell Preservation Upstream of Single Cell RNA Sequencing

Single cell RNA sequencing (scRNA-seq) is a revolutionary technique to identify cell types and their molecular phenotype in heterogeneous biological specimens. ScRNA-seq typically requires fresh, high quality single cell suspensions that are processed immediately to preserve their molecular profiles. This presents a challenge for samples with long preparation times and prevents collection at remote sites lacking the required instrumentation for sample processing. Recently, several commercial assays have been released that enable sample preservation at the time of collection either via fixation or cryopreservation, allowing for sample processing to occur months after the initial collection. The Association of Biomolecular Research Facilities (ABRF) DNA Sequencing (DSRG) and Genomics Bioinformatics (GBiRG) Research Groups have undertaken a cross-platform, multi-site study to assess the performance and reproducibility of three platforms: a) 10x Genomics FLEX, b) Parse Bioscience Evercode WT v2 and c) Honeycomb Bio HIVE. Total leukocytes were isolated from a single healthy individual using the EasySep RBC depletion reagent. Cells were then characterized by collecting a 21-color flow cytometry dataset for reference and the remaining material was used for scRNA-seq procedures where different sites then processed either the fixed or cryopreserved cells for each method. We evaluated performance of each method across traditional scRNA-seq quality control metrics and analysis applications, including gene/transcript detection sensitivity, cell type discovery and annotation, and differential expression. We demonstrate that data from the methods tested can be effectively integrated and produce concordant results with regard to cell type annotation and relative abundance, though we observe platform-specific differences in the expression of a subset of genes. Preservation-based methods also show better retention of fragile granulocyte populations compared with fresh samples processed using the 10x 3 workflow. The improvements to preservation methods are changing the way research is conducted and our thorough investigation into the performance of each method provides a valuable resource to help scientists determine the most appropriate single cell preservation workflow given their sample collection logistics and laboratory infrastructure constraints.

genomics↗

bioRxiv2025

Cell fate specification during respiratory development requires ARID1A-containing canonical BAF complex activity

Development of the mammalian lung requires formation of a definitive lung bud from the foregut endoderm, branching morphogenesis, specification of a proximal to distal axis, and differentiation of extensive specialized airway and alveolar epithelial lineages. These steps require coordinated temporal and regional gene expression to define the lung epithelium in preparation for the first breath at birth. While the transcriptional and signaling regulators required for lung development are increasingly well known, the key epigenetic complexes that interact with lineage transcription factors for cell-specific control of gene expression remain to be defined. Here, we identify a key role for the canonical BAF complex, an ATP-dependent chromatin remodeling complex, during lung epithelial development. Loss of canonical BAF activity throughout the foregut endoderm leads to complete failure of lung formation, and selective deletion of a single key subunit, ARID1A, leads to failure of proximal-distal axis specification, with dilated airway-like structures lined by ectopic basal cells found throughout the distal lung, and failure of specification of alveolar type 1 (AT1) cells in the distal saccular epithelium. In place of AT1 cells, we identified a highly proliferative epithelial cell state defined by joint activation of YAP and WNT signaling and loss of BMP signaling response, leading to increased proliferation and failure of appropriate epithelial differentiation. These changes resulted in secondary failure of mesenchymal and endothelial specification, leading to broad loss of patterning of the distal lung further disrupting peripheral lung morphogenesis. Using embryonic lung organoids, we demonstrate that exogenous BMP4 signaling is sufficient to rescue AT1 and AT2 cell differentiation in ARID1A-null epithelium, while WNT and YAP signaling require functional BAF complex. Together, these data demonstrate a requirement for the BAF complex for lung formation, proximal-distal patterning, and cell fate acquisition, and reveal surprising differential specificity between signaling pathways during lung development. One-Sentence SummaryFunctional BAF complex containing ARID1A is required for lung development.

developmental biology↗

bioRxiv2025

Targeted genomic surveillance of insecticide resistance in African malaria vectors

The emergence of insecticide resistance is threatening the efforts of malaria control programmes, which rely heavily on a limited arsenal of insecticidal tools, such as insecticide-treated bed nets. Importantly, genomic surveillance of malaria vectors can provide critical, policy-relevant insights into the presence and evolution of insecticide resistance, allowing us to maintain and extend the shelf life of these interventions. Yet the complex genetic architecture of resistance, combined with resource constraints in malaria-endemic settings, have thus far precluded the widespread use of genomics in routine surveillance. Meanwhile, stakeholders in sub-Saharan Africa are moving towards locally driven, decentralised generation of genomic data, underscoring the need for standardised and robust genomics workflows. To address this need, we demonstrate an approach to targeted genomic surveillance in Anopheles gambiae s.l with Illumina sequencing. We target 90 genomic loci in the Anopheles gambiae s.l genome, including 55 resistance-associated mutations and 35 ancestry informative markers. This protocol is coupled with advanced, automated software for accurate and reproducible variant analysis. We are able to elucidate population structure and ancestry in our cohorts and accurately identify most species in the An. gambiae species complex. We report frequencies of variants at insecticide-resistance loci and explore the continued evolution of the pyrethroid target site, the Voltage-gated sodium channel. Applying the platform to a recently established colony of field-caught resistant mosquitoes (Siaya, Kenya), we identified seven independent resistance-associated variants contributing to reduced efficacy of insecticide-treated nets in East Africa. Additionally, we leverage a machine learning algorithm (XGBoost) to demonstrate the possibility of predicting bioassay mortality using genotypes alone. This achieved high accuracy (75%), demonstrating the potential of targeted genomics to predictively monitor insecticide resistance. Together these tools provide a practical, scalable solution for resistance monitoring while advancing the goal of building local genomic surveillance capacity in sub-Saharan Africa.

genomics↗

bioRxiv2024

Vitamin B12 partially rescues embryonic cell migration defects in C. elegans ephrin mutants by improving both propionic acid breakdown and one-carbon cycle metabolic pathways

Successful cell migration followed by tissue fusion is required for organogenesis in a number of tissues, many of which are susceptible to gene-environment interactions that can result in congenital anomalies. In C. elegans embryogenesis, one such event is the closure of the ventral cleft, which must occur as the first step in morphogenesis, otherwise embryos arrest; this process depends on ephrin signaling, but no single gene mutation is fully penetrant embryonic lethal. We exposed hermaphrodites mutant for vab-1, the C. elegans ephrin receptor, to various environmental conditions and found that vitamin B12 supplementation could partially rescue the embryonic lethality of multiple alleles from 40% survival to 63% survival. Analysis of vab-1 mutant phenotypes showed that vitamin B12 reduced the frequency of ventral cleft closure failure by promoting more normal cell positions and increased migration by the precursor cells of those that adhere to close the cleft. We found that vitamin B12 partially rescued the embryonic lethality of other ephrin pathway mutants, but not mutants that have ventral cleft defects due to cell adhesion or cell fate defects. We found that rescue by vitamin B12 depends on its functions in both mitochondrial propionic acid breakdown and the one-carbon cycle, and that antioxidant treatment can also partially rescue ephrin pathway mutants. These results are distinct from the larval response to vitamin B12, which depends only on the one-carbon cycle, emphasizing the unique metabolism of embryos and particularly the metabolic needs of migrating cells. Overall, our findings highlight the C. elegans embryo a model system to investigate gene-environment interactions and developmental metabolism.

developmental biology↗

bioRxiv2024

Solanum pan-genomics and pan-genetics reveal paralogs as contingencies in crop engineering

Pan-genomics and genome editing technologies are revolutionizing the breeding of globally cultivated crops. A transformative opportunity lies in the reciprocal exchange of genotype-to-phenotype knowledge of agricultural traits between these major crops and hundreds of locally cultivated indigenous crops, thereby enhancing the diversity and resilience of our food system. However, species-specific genetic variants and their interactions with desired natural or engineered mutations pose barriers to achieving predictable phenotypic effects, even between closely related crops or genotypes. Here, by establishing a pan-genome of the crop-rich genus Solanum and integrating functional genomics and genetics, we show that gene duplication and subsequent paralog diversification are a major obstacle to genotype-phenotype predictability. Despite broad conservation of gene macrosynteny among chromosome-scale references for 22 species, including 13 indigenous crops, hundreds of global and lineage-specific gene duplications exhibited dynamic evolutionary trajectories in paralog sequence, expression, and function, including among members of key domestication gene families. Extending our pan-genome with 10 cultivars of African eggplant and leveraging quantitative genetics and genome editing, we uncovered an intricate history of paralog emergence and evolution within this indigenous crop. The loss of an ancient redundant paralog of the classical regulator of stem cell proliferation and fruit organ number, CLAVATA3 (CLV3), was compensated by a lineage-specific tandem duplication. Subsequent pseudogenization of the derived copy followed by a cultivar-specific structural variant resulted in a single fused functional copy of CLV3 that modifies locule number alongside a newly identified gene controlling the same trait. Our findings demonstrate that paralog diversifications over short evolutionary periods are critical yet underexplored contingencies in trait evolvability and independent crop domestication histories. Unraveling these contingencies is crucial for translating genotype-to-phenotype relationships across related species.

genetics↗

bioRxiv2024

Exploring the genetic and epigenetic underpinnings of early-onset cancers: Variant prioritization for long read whole genome sequencing from family cancer pedigrees

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 de novo 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.

genomics↗

bioRxiv2024

Gapless assembly of complete human and plant chromosomes using only nanopore sequencing

The combination of ultra-long Oxford Nanopore (ONT) sequencing reads with long, accurate PacBio HiFi reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, "telomere-to-telomere" genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT "Duplex" sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely-studied genomes: human HG002, Solanum lycopersicum Heinz 1706 (tomato), and Zea mays B73 (maize). For the diploid, heterozygous HG002 genome, we also used "Pore-C chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the ultra-long reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and has the potential to provide a single-instrument solution for the reconstruction of complete genomes.

bioinformatics↗

bioRxiv2023

The genome of the Wollemi pine, a critically endangered living fossil unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

genomics↗

bioRxiv2023

Using deep long-read RNAseq in Alzheimer's disease brain to assess clinical relevance of RNA isoform diversity

Due to alternative splicing, human protein-coding genes average over eight RNA isoforms, resulting in nearly four distinct protein coding sequences per gene. Long-read RNAseq (IsoSeq) enables more accurate quantification of isoforms, shedding light on their specific roles. To assess the medical relevance of measuring RNA isoform expression, we sequenced 12 aged human frontal cortices (6 Alzheimers disease cases and 6 controls; 50% female) using one Oxford Nanopore PromethION flow cell per sample. Our study uncovered 53 new high-confidence RNA isoforms in medically relevant genes, including several where the new isoform was one of the most highly expressed for that gene. Specific examples include WDR4 (61%; microcephaly), MYL3 (44%; hypertrophic cardiomyopathy), and MTHFS (25%; major depression, schizophrenia, bipolar disorder). Other notable genes with new high-confidence isoforms include CPLX2 (10%; schizophrenia, epilepsy) and MAOB (9%; targeted for Parkinsons disease treatment). We identified 1,917 medically relevant genes expressing multiple isoforms in human frontal cortex, where 1,018 had multiple isoforms with different protein coding sequences, demonstrating the need to better understand how individual isoforms from a single gene body are involved in human health and disease, if at all. Exactly 98 of the 1,917 genes are implicated in brain-related diseases, including Alzheimers disease genes such as APP (A{beta} precursor protein; five), MAPT (tau protein; four), and BIN1 (eight). As proof of concept, we also found 99 differentially expressed RNA isoforms between Alzheimers cases and controls, despite the genes themselves not exhibiting differential expression. Our findings highlight the significant knowledge gaps in RNA isoform diversity and their medical relevance. Deep long-read RNA sequencing will be necessary going forward to fully comprehend the medical relevance of individual isoforms for a "single" gene.

genomics↗

bioRxiv2023

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

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 by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mtb genes. We explored the role of codon pair bias by recoding Mtb genes (rpoB, mmpL3, 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. IMPORTANCEMycobacterium 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 the first report that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

molecular biology↗

bioRxiv2022

Low-level repressive histone marks fine-tune stemness gene transcription in neural stem cells

Coordinated regulation of stemness gene activity by transcriptional and translational mechanisms poise stem cells for a timely cell-state transition during differentiation. Although important for all stemness-to-differentiation transitions, mechanistic understanding of the fine-tuning of stemness gene transcription is lacking due to the compensatory effect of translational control. We used intermediate neural progenitor (INP) identity commitment to define the mechanisms that fine-tune stemness gene transcription in fly neural stem cells (neuroblasts). We demonstrate that the transcription factor FruitlessC (FruC) binds cis-regulatory elements of most genes uniquely transcribed in neuroblasts. Loss of fruC function alone has no effect on INP commitment but drives INP dedifferentiation when translational control is reduced. FruC negatively regulates gene expression by promoting low-level enrichment of the repressive histone mark H3K27me3 in gene cis-regulatory regions. Identical to fruC loss-of-function, reducing Polycomb Repressive Complex 2 activity increases stemness gene activity. We propose low-level H3K27me3 enrichment fine-tunes stemness gene transcription in stem cells, a mechanism likely conserved from flies to humans.

developmental biology↗

bioRxiv2022

Intratumoral heterogeneity and clonal evolution induced by HPV integration

The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive cancers, but the consequences for chromosomal integrity are unknown. Continuous long-read sequencing of oropharyngeal cancers and cancer cell lines revealed a unique form of structural variation, i.e., heterocateny, characterized by diverse, interrelated, and repetitive patterns of concatemerized virus and host DNA segments within a cancer. Unique breakpoint sequences shared across structural variants facilitated stepwise reconstruction of their evolution from a common molecular ancestor. This analysis revealed that virus and virus-host concatemers are unstable and, upon insertion into and excision from chromosomes, facilitate capture, amplification, and recombination of host DNA and chromosomal rearrangements. Evidence of heterocateny was detected in extrachromosomal and intrachromosomal DNA. The data indicate that heterocateny is driven by the dynamic, aberrant replication and recombination of an oncogenic DNA virus, thereby extending known consequences of HPV integration to include promotion of intratumoral heterogeneity and clonal evolution.

cancer biology↗

bioRxiv2022

Candidate effector proteins from the maize tar spot pathogen Phyllachora maydis localize to diverse plant cell compartments

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmECs) proteins remain unknown and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super Yellow Fluorescent Protein (sYFP)-tagged P. maydis effector candidate proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane while an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest effector candidate proteins from P. maydis target diverse cellular organelles and may thus provide valuable insights into their putative functions as well as host processes potentially manipulated by this fungal pathogen.

plant biology↗

bioRxiv2022

ACME: an Affinity-based Cas9 Mediated Enrichment method for targeted nanopore sequencing

Targeted sequencing significantly improves accuracy and coverage and aids in providing the depth necessary to detect rare alleles in a heterogenous population of cells. Until the introduction of nanopore Cas9 Targeted-Sequencing (nCATS), a lack of efficient long-read compatible targeting techniques made it difficult to study specific regions of interest on long-read platforms. Existing nCATS-based strategies are currently limited by the per molecule target lengths capturable (<30kb), requiring several Cas9 guides to tile across larger regions of interest, ultimately reducing the number of targets that can be surveyed per reaction. Also, longer read lengths help reduce mapping errors, making it more likely that complex structural rearrangements can be resolved. Absence of a background reduction step in nCATS also increases the competition between non-target and target fragments in the sequencing pool for pore occupancy, decreasing the overall percentage of on-target reads. To address this, we introduce ACME - an Affinity-based Cas9-Mediated Enrichment method - that helps reduce background reads, increasing on-target coverage and size of target regions that can be spanned with single reads to 100kb. ACME uses a HisTag-based isolation and pulldown of Cas-9 bound non-target reads, reducing the background noise in sequencing. We designed a panel of guide RNAs targeting 10 genes to enrich for specific regions of the cancer genome and tested them in two breast cell lines - MCF 10A and SK-BR-3. These gene targets spanned different size ranges (10kb to 150kb) allowing us to identify the largest target sizes that could be optimally captured by single molecules spanning the entire region. When compared with using just nCATS, the ACME method for background reduction increased the overall coverage across the entire length of all targets by 2-fold to 25-fold. By using ACME to eliminate smaller competing non-targets from the sequencing library, we saw a 3- to 7-fold increase in the number of reads spanning 100% of the gene targets when compared to nCATS. For one of our larger targets, BRCA2, we observed >60-fold target enrichment, close to 70x coverage, and 3-20 reads spanning the entire 95kb target. We observed an increase in enrichment, depth, and number of whole gene spanning reads for other genes on the panel as well across both cell lines, with enrichment as high as 4000-fold for some genes. Furthermore, ACME identified all SVs previously called within our targets by ONT and PacBio whole genome sequencing and performed on par with these platforms for SNP detection when compared with Illumina short-read whole genome sequencing.

genomics↗

bioRxiv2021

Fly Cell Atlas: a single-cell transcriptomic atlas of the adult fruit fly

The ability to obtain single cell transcriptomes for stable cell types and dynamic cell states is ushering in a new era for biology. We created the Tabula Drosophilae, a single cell atlas of the adult fruit fly which includes 580k cells from 15 individually dissected sexed tissues as well as the entire head and body. Over 100 researchers from the fly community contributed annotations to >250 distinct cell types across all tissues. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types that are shared between tissues, such as blood and muscle cells, allowed the discovery of rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the entire Drosophila community and serves as a comprehensive reference to study genetic perturbations and disease models at single cell resolution.

genomics↗

bioRxiv2020

High resolution copy number inference in cancer using short-molecule nanopore sequencing

Genome copy number is an important source of genetic variation in health and disease. In cancer, clinically actionable Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in terms of the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms. This represents a challenge for applications that require high read counts such as CNA inference. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that sequencing short DNA molecules reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for the sequencing of relatively short DNA molecules on nanopore devices with applications in research and medicine, that include but are not limited to, CNAs.

genomics↗