Capture Sequencing Research Articles

CSpredR: A Multi-Site mRNA Subcellular Localization Prediction Method Based on Fusion Encoding and Hybrid Neural Networks

Current research widely acknowledges that the subcellular localization of mRNA is crucial for understanding its biological functions. However, current methods for mRNA subcellular localization based on k-mer frequency features may overlook the sequential information of the sequence, and a single encoding method may not adequately extract the sequence’s features. This paper proposes a novel deep learning prediction method, CSpredR, specifically designed for predicting the subcellular localization of multi-site mRNAs. Unlike previous methods, CSpredR first employs k-mer to tokenize the mRNA sequences, then converts the tokenized sequences into de Bruijn graphs, thereby enabling a more precise capture of the structural information within the sequences. To mitigate the impact of lost sequential information and better capture sequence features, we combine word2vec and fasttext models to extract the features of each node in the graph and retain the sequence order. They can encode the k-mer units in the sequence into word vectors, thus serving as the node feature vectors of the graph. In this way, each node in the graph is assigned a feature vector containing rich semantic information. Subsequently, we utilize multi-scale convolutional neural networks and bidirectional long short-term memory networks to capture sequence features, respectively, and fuse the results as input for a multi-head attention mechanism model. The information from these heads is integrated into the node representations, and finally, the attention-processed data are fed into an MLP (Multi-Layer Perceptron) for prediction tasks. Extensive experiments reveal that CSpredR achieves a 2% improvement over the best existing predictors, offering a more effective tool for mRNA subcellular localization prediction.

DNA Origami Framework-Based Spatial Nanochip for Circular ssDNA Assembly and Data Storage.

A 3D DNA spatial chip (DSC) based on an icosahedral DNA origami framework is introduced to construct customized circular single-stranded DNA (c-ssDNA) for data storage. Within the confined space of the DSC, thirty addressable location sequences extending from the framework edges are available for designing circular paths and directing the assembly of a series of information oligonucleotides for efficient ligation. This strategy is verified by constructing c-ssDNAs from up to 15 fragments toencode two poems (800 and 860 nucleotides). Using orthogonal location sites, both poems are simultaneously assembled within a single DSC and read out together. Rolling circle amplification (RCA) and nanopore sequencing enable complete retrieval of all the above data files. The DSCs with distinct fluorescent labels and capture sequences are further functionalized on their outer surfaces, allowing magnetic bead-based retrieval and rapid identification of specific datasets from a mixture. Moreover, the DSCs maintain data integrity after storage under various conditions. These findings demonstrate that the 3D DNA spatial chip provides an efficient approach for assembling long c-ssDNA for data storage, addressing limitations by reducing redundancy, enhancing stability, and enabling multiplexed storage and retrieval.

Cutting-edge tools for unveiling the dynamics of plasmid-host interactions.

The plasmid-mediated transfer of antibiotic resistance genes (ARGs) in complex microbiomes presents a significant global health challenge. This review examines recent technological advancements that have enabled us to move beyond the limitations of culture-dependent detection of conjugation and have enhanced our ability to track and understand the movement of ARGs in real-world scenarios. We critically assess the applications of single-cell sequencing, fluorescence-based techniques and advanced high-throughput chromatin conformation capture (Hi-C) approaches in elucidating plasmid-host interactions at unprecedented resolution. We also evaluate emerging techniques such as CRISPR-based phage engineering and discuss their potential for developing targeted strategies to curb ARG dissemination. Emerging data derived from these technologies have challenged our previous paradigms on plasmid-host compatibility and an awareness of an emerging uncharted realm for ARGs.

Quantitative Analysis of Hepatitis D Virus Using gRNA-Sensitive Semiconducting Polymer Dots.

Hepatitis D virus (HDV) significantly influences the progression of liver diseases. Through clinical observations and database analyses, it has been established that patients coinfected with HDV and hepatitis B virus (HBV) experience accelerated progression toward cirrhosis, hepatocellular carcinoma (HCC), and liver failure compared to those infected solely with HBV. A higher viral load correlates with increased replicative activity, enhanced infectivity, and more severe disease manifestations. In this study, we use HDV gRNA-sensitive semiconducting polymer dots (Pdots) as the nanoprobes for the quantitative analysis of HDV copy number variations. The surface of the Pdots is engineered with a clamp design that includes a pair of reporter sequences, protection sequences, and capture sequences tailored to the conserved sequence length of the HDV genome. The capture sequence, comprising leading and trailing chains, specifically binds to the gRNA of the target virus. The protection sequence shields the Pdots from external interference, while the reporter sequence detects the presence of target gRNA through the degradation of fluorescent dye Cy5.5dt. We demonstrate the effectiveness of this assay in a stably transfected cell line derived from HepG2-HDV cells and its translational application in clinical samples from patients. Additionally, this nanobiosensor can accurately detect gRNA at femtomolar (fM) levels, a sensitivity unachievable by previously reported methods. This novel virus quantification system offers significant potential for clinical and virological applications, enhancing screening, early diagnosis, and personalized treatment strategies.

Short INDELs and SNPs as markers of evolutionary processes in hybrid zones.

Polymorphic short insertions and deletions (INDELs ≤ 50bp) are abundant, although less common than single nucleotide polymorphisms (SNPs). Evidence from model organisms shows INDELs to be more strongly influenced by purifying selection than SNPs. Partly for this reason, INDELs are rarely used as markers for demographic processes or to detect divergent selection. Here, we compared INDELs and SNPs in the intertidal snail Littorina saxatilis, focusing on hybrid zones between ecotypes, in order to test the utility of INDELs in the detection of divergent selection. We computed INDEL and SNP site frequency spectra (SFS) using capture sequencing data. We assessed the impact of divergent selection by analysing allele frequency clines across habitat boundaries. We also examined the influence of GC-biased gene conversion because it may be confounded with signatures of selection. We show evidence that short INDELs are affected more by purifying selection than SNPs, but part of the observed SFS difference can be attributed to GC-biased gene conversion. We did not find a difference in the impact of divergent selection between short INDELs and SNPs. Short INDELs and SNPs were similarly distributed across the genome and so are likely to respond to indirect selection in the same way. A few regions likely affected by divergent selection were revealed by INDELs and not by SNPs. Short INDELs can be useful (additional) genetic markers helping to identify genomic regions important for adaptation and population divergence.

A thorny tale: The origin and diversification of Cirsium (Compositae).

Widely distributed plant genera offer insights into biogeographic processes and biodiversity. The Carduus-Cirsium group, with over 600 species in eight genera, is diverse across the Holarctic regions, especially in the Mediterranean Basin, Southwest Asia, Japan, and North America. Despite this diversity, evolutionary and biogeographic processes within the group, particularly for the genus Cirsium, remain underexplored. This study examines the biogeographic history and diversification of the group, focusing on Cirsium, using the largest molecular dataset for the group (299 plants from 251 taxa). Phylogenomic analyses based on 350 nuclear loci, derived from target capture sequencing, revealed highly resolved and consistent phylogenetic trees, with some incongruences likely due to hybridization and incomplete lineage sorting. Ancestral range estimations suggest that the Carduus-Cirsium group originated during the Late Miocene in the Western Palearctic, particularly in the Mediterranean, Eastern Europe, or Southwest Asia. A key dispersal event to tropical eastern Africa around 10.7 million years ago led to the genera Afrocarduus and Afrocirsium, which later diversified in the Afromontane region. The two subgenera of Cirsium-Lophiolepis and Cirsium-began diversifying around 7.2-7.3 million years ago in the Western Palearctic. During the Early Pliocene, diversification rates increased, with both subgenera dispersing to Southwest Asia, where extensive in situ diversification occurred. Rapid radiations in North America and Japan during the Pleistocene were triggered by jump-dispersals events from Asia, likely driven by geographic isolation and ecological specialization. This added further layers of complexity to the already challenging taxonomic classification of Cirsium.Keywords: Biogeography; Carduinae; Cirsium; Diversification; North Hemisphere; Target-enrichment; Taxonomy.

Impact of structural variations and genome partitioning on bread wheat hybrid performance.

The agronomical interest of hybrid wheat has long been a matter of debate. Compared to maize where hybrids have been successfully grown for decades, the mixed results obtained in wheat have been attributed at least partially to the lack of heterotic groups. The wheat genome is known to be strongly partitioned and characterized by numerous presence/absence variations and alien introgressions which have not been thoroughly considered in hybrid breeding. The objective was to investigate the relationships between hybrid performance and genomic diversity. For this, we characterized a set of 124 hybrids as well as their 19 female and 16 male parents. Phenotyping for yield and yield components was conducted during two years in three locations. Parental lines were genotyped using a 410K SNP array as well as through sequence capture of roughly 200,000 loci. This led to the identification of 180 structural variations including presence-absence variations and alien introgressions. Twenty-six of them were associated to hybrid performance through either additivity or dominance effects. While no correlation was observed at the whole genome level, the genetic distance for 25 genomic regions resulting from the structural and functional partitioning of the chromosomes shown positive or negative correlation with agronomic traits including yield. Large introgressions, like the Aegilops ventricosa 2NS-2AS translocation, can correspond to entire chromosomal regions, such as the R1 region, with an impact on yield. Our results suggest hybrid breeding should consider both structural variations and chromosome partitioning rather than maximizing whole-genome genetic distance, and according to genomic regions to combine homozygosity and heterozygosity.

A Post-Mortem Molecular Damage Profile in the Ancient Human Mitochondrial DNA.

Mitochondrial DNA (mtDNA) analysis is crucial for understanding human population structure and genetic diversity. However, post-mortem DNA damage poses challenges, that make analysis difficult. DNA preservation is affected by environmental conditions which, among other factors, complicates the differentiation of endogenous variants from artefacts in ancient mtDNA mix profiles. This study aims to develop a molecular damage profile for ancient mtDNA that can become a useful tool in analysing mtDNA from ancient remains. A dataset of 427 whole genomes or capture of mtDNA sequences from individuals representing different historical periods and climatic regions was compiled from the ENA database. Present-day and UDG-treated ancient samples were also included and used to establish levels of damaged reads. Results indicated that samples from cold regions exhibited the lowest percentage of damaged reads, followed by arid, cold, tropical and temperate regions, with significant differences observed between cold and temperate regions. A global damage profile was generated, identifying 2933 positions (25% of the positions considered) with damage in more than 23.8% of the samples analysed, deemed as damage hotspots. Notably, 2856 of these hotspots had never been reported as damage or mutational hotspots, or heteroplasmic positions. Damage hotspot frequency by position was slightly higher in the non-coding region compared with the coding region. In conclusion, this study provides a molecular damage profile for ancient mtDNA analysis that is expected to be a valuable tool in the interpretation of mtDNA variation in ancient samples.

Adding Virome Capture Metagenomic Sequencing to Conventional Laboratory Testing Increases Unknown Fever Etiology Determination in Bamako, Mali

Unexplained fever poses significant diagnostic challenges in resource-limited settings like Bamako, Mali, where overlapping endemic diseases include malaria, HIV/AIDS, yellow fever, typhoid, and others. This study aimed to elucidate the infectious etiologies of acute febrile illnesses in this context. Acute febrile patients of any age were enrolled after informed consent or assent. Baseline clinical and demographic data were collected, and samples were analyzed by using rapid diagnostic tests, reverse transcriptase polymerase chain reaction, ELISA, and virus-targeted metagenomic sequencing (virome capture sequencing platform for vertebrate viruses [VirCapSeq-VERT]). Among 108 enrolled subjects, most were male (51.9%) and under 15 years old (56.5%). Measles virus was identified in 39.8% of cases, primarily among children. Other findings included Plasmodium spp. (12%), Salmonella spp. (13%), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; 8.7%). The virome capture sequencing platform for vertebrate viruses was used for 101 subjects, corroborating many routine test results and identifying additional cases of measles virus (1), SARS-CoV-2 (5), and numerous other agents. Notably, nearly all subjects showed evidence of herpesviruses (90%) and anelloviruses (98%). Hemorrhagic fever viruses were not observed. With the inclusion of VirCapSeq-VERT, identifiable pathogens were found in 79.6% of cases, leaving 20.4% without a clear etiology. The identification of more than one concurrent pathogen was common (41.5%). Integrating metagenomic sequencing with routine laboratory diagnostic testing enhances the detection of pathogens in acute febrile illnesses, highlighting its potential value in identifying infectious etiologies in resource-limited settings.

HIV-SEQ REVEALS GLOBAL HOST GENE EXPRESSION DIFFERENCES BETWEEN HIV-TRANSCRIBING CELLS FROM VIREMIC AND SUPPRESSED PEOPLE WITH HIV.

"Active" reservoir cells transcribing HIV can perpetuate chronic inflammation in virally suppressed people with HIV (PWH) and likely contribute to viral rebound after antiretroviral therapy (ART) interruption, so they represent an important target for new therapies. These cells, however, are difficult to study using single-cell RNA-seq (scRNA-seq) due to their low frequency and low levels of HIV transcripts, which are usually not polyadenylated. Here, we developed "HIV-seq" to enable more efficient capture of HIV transcripts - including non-polyadenylated ones - for scRNA-seq analysis of cells from PWH. By spiking in a set of custom-designed capture sequences targeting conserved regions of the HIV genome during scRNA-seq, we increased our ability to find HIV RNA+ cells from PWH by up to 44%. Implementing HIV-seq in conjunction with surface phenotyping by CITE-seq on paired blood specimens from PWH before vs. after ART suppression, we found that HIV RNA+ cells were enriched among T effector memory (Tem) cells during both viremia and ART suppression, but exhibited a cytotoxic signature during viremia only. By contrast, HIV RNA+ cells from the ART-suppressed timepoints exhibited a distinct anti-inflammatory signature involving elevated TGF-β and diminished IFN signaling. Overall, these findings demonstrate that active reservoir cells exhibit transcriptional features distinct from HIV RNA+ cells during viremia, and underscore HIV-seq as a useful tool to better understand the mechanisms by which HIV-transcribing cells can persist during ART.

Chromosome-level genome assembly and annotation of a sea toad (Chaunax sp.)

The sea toad genus Chaunax is a group of small benthic fishes that predominantly inhabiting the deep seas of the Atlantic, Indian, and Pacific Oceans. Although they have the potential to make excellent systems for studies of evolutionary adaptation to deep-sea environments, genomic research on Chaunax has been hindered by a scarcity of high-quality genomic resources. We present a chromosome-scale genome assembly of a Chaunax specimen generated using PacBio long-read sequencing and high-throughput chromosome conformation capture technology. The size of the assembled genome was 706.94 Mb, with a contig N50 of 15.24 Mb and scaffold N50 of 29.42 Mb. Approximately 96.11% of assembled sequences were anchored and oriented onto 24 pseudo-chromosomes. The genome contained 213.47 Mb repetitive sequences, 25,280 protein-coding genes, and 5,090 non-coding RNAs. The high ratio of complete BUSCO genes (97.20%) indicates high quality of genome assembly. The chromosomal-level reference genome of Chaunax sp. provides a preliminary molecular basis for understanding deep-sea adaptation and phenotypic evolution as well as an important reference for whole-genome sequencing of related species.

High quality genome assembly and annotation (v1) of the eukaryotic freshwater microalga Coccomyxa elongata SAG 216-3b.

Unicellular green algae of the genus Coccomyxa are recognized for their worldwide distribution and ecological versatility. Coccomyxa elongata is a freshwater species of the Coccomyxa simplex clade, which also includes lichen symbionts. To facilitate future molecular and phylogenomic studies of this versatile clade of algae, we generated a high-quality genome assembly for Coccomyxa elongata Chodat & Jaag SAG 216-3b within the framework of the Biodiversity Genomics Center Cologne (BioC2) initiative. A combination of long-read PacBio HiFi and Oxford Nanopore Technologies with chromatin conformation capture (Hi-C) sequencing led to the assembly of the genome into 21 scaffolds with a total length of 51.4 Mb and an N50 of 2.8 Mb. Nineteen of the scaffolds represent highly complete nuclear chromosomes delimited by telomeric repeats, while the two additional scaffolds represent the mitochondrial and plastid genomes. Transcriptome-guided gene annotation resulted in the identification of 14,811 protein-coding genes, of which 61% have annotated PFAM domains and 841 are predicted to be secreted. BUSCO analysis against the Chlorophyta database identified a total of 1,494 (98.4 %) complete gene models, suggesting a highly complete genome annotation.

Improved precision, sensitivity, and adaptability of ordered two-template relay cDNA library preparation for RNA sequencing.

Sequencing RNAs that are biologically processed or degraded to less than ∼100 nt typically involves multistep, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced ordered two-template relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced nonproductive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 pg (<1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from unwanted adapter-only side products. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or posttranscriptional modifications.

Chromosome-level genome assembly of Iodes seguinii and its metabonomic implications for rheumatoid arthritis treatment.

Iodes seguinii is a woody vine known for its potential therapeutic applications in treating rheumatoid arthritis (RA) due to its rich bioactive components. Here, we achieved the first chromosome-level assembly of the nuclear genome of I. seguinii using PacBio HiFi and chromatin conformation capture (Hi-C) sequencing data. The initial assembly with PacBio data produced contigs with an N50 length of 9.71 Mb, and Hi-C data anchored these contigs into 13 chromosomes, achieving a total length of 273.58 Mb, closely matching the estimated genome size. Quality assessments, including BUSCO, long terminal repeat assembly index, transcriptome mapping rates, and sequencing coverage, confirmed the high quality, completeness, and continuity of the assembly, identifying 115.28 Mb of repetitive sequences, 1062 RNA genes, and 25,270 protein-coding genes. Additionally, we assembled and annotated the 150,599bp chloroplast genome using Illumina sequencing data, containing 121 genes including key DNA barcodes, with maturase K (matK) proving effective for species identification. Phylogenetic analysis positioned I. seguinii at the base of the Lamiales clade, identifying significant gene family expansions and contractions, particularly related to secondary metabolite synthesis and DNA damage repair. Metabolite analysis identified 84 active components in I. seguinii, including the discovery of luteolin, with 119 targets predicted for RA treatment, including core targets like AKT1, toll-like receptor 4 (TLR4), epidermal growth factor receptor (EGFR), tumor necrosis factor (TNF), TP53, NFKB1, janus kinase 2 (JAK2), BCL2, mitogen-activated protein kinase 1 (MAPK1), and spleen-associated tyrosine kinase (SYK). Key active components such as flavonoids and polyphenols with anti-inflammatory activities were highlighted. The discovery of luteolin, in particular, underscores its potential therapeutic role. These findings provide a valuable genomic resource and a scientific basis for the development and application of I. seguinii, addressing the genomic gap in the genus Iodes and the order Icacinales and underscoring the need for further research in genomics, transcriptomics, and metabolomics to fully explore its potential.

Chromosome-level genome assembly of the Suminoe oyster Crassostrea ariakensis in south China

The Suminoe oyster Crassostrea ariakensis (Fujita, 1913) is one of the most important ecological and fishery bivalve mollusks with a worldwide distribution. Here, we reported an improved high-quality chromosomal-level genome assembly of C. ariakensis inhabiting the South China Sea, using Nanopore technology, Illumina sequencing, and high-throughput chromosomal conformation capture analysis. The assembled genome size is 631.73 Mb, with contig N50 length of 5.36 Mb and scaffold N50 length of 61.15 Mb, and is assigned to 10 chromosomes. A total of 29,357 protein-coding genes are predicted, 96.68% of which are functionally annotated. The genome contains 347.11 Mb (54.94%) of repetitive elements and 1130 noncoding RNAs. This improved genome assembly of south C. ariakensis is an important resource for understanding oyster diversity and evolution, and provides insights into genetic improvement, protection and management of oyster resource.

Development of a 50K SNP array for whole-genome analysis and its application in the genetic localization of eggplant (Solanum melongena L.) fruit shape.

Current eggplant variety breeding is still mainly based on conventional methods, and there remains a lack of effective molecular breeding systems for complex traits controlled by multiple genes, such as yield and quality. To accelerate the research progress of eggplant genetics and molecular breeding, it is necessary to implement a genome-based breeding strategy. Therefore, in this study, a SNP array containing 50K liquid-phase probes was designed on the basis of the resequencing data of 577 eggplants. The developed 50K liquid-phase probes were used to perform targeted capture sequencing on 12 eggplant lines, and the efficiency of probe capture exceeded 99.25%. Principal component, phylogenetic, and population structure analyses divided the 577 eggplants into 7 subgroups, and statistical analysis was performed on the fruit shape and color of the materials in the different subgroups. Further analysis of the geographical distribution of 428 Chinese eggplant materials revealed that the geographical regions of different subgroups were similar. The 50K SNP liquid-phase array was used to perform bulked- segregant analysis combined with whole-genome resequencing (BSA-seq) of fruit shape in the F2 population, which consisted of 1435 lines constructed with E421 as the maternal parent and 145 as the paternal parent. The BSA-seq data were located in the 78444173-84449348 interval on chromosome 3, with a size of 6 Mb, which was narrowed to 712.6 kb through fine mapping. Further sequence alignment and expression analysis revealed SmIQD14 as a candidate gene controlling eggplant fruit shape. The 50K SNP liquid-phase array can be widely used in future eggplant molecular breeding research.

Surveillance and agnostic capture sequencing of samples from individuals with rash-associated illness in Mali indicates regional transmission of measles virus from West and Central Africa

Measles is vaccine-preventable extremely contagious disease caused by the measles virus. High vaccination coverage is needed to prevent outbreaks of disease. Although molecular surveillance of measles is critical to characterize outbreaks and track viral evolution, few whole-genome sequences of measles virus from West Africa are available despite continual outbreaks in the region. Using VirCapSeq-VERT, an enhanced and comprehensive metagenomic sequencing technique that allows for simultaneous identification of all vertebrate viruses, 23 wild-type near-complete genomes of measles virus from across Mali were obtained from samples collected between January 2012 to October 2022. Other febrile rash illnesses were also identified by VirCapSeq-VERT, demonstrating the advantage of using broad detection agnostic methods when the clinical diagnosis is unclear. Whereas one measles virus sequence was consistent with measles vaccine-associated rash illness (VARI), the remaining 38 were classified within the B3.1 genotype. Broad surveillance throughout Mali reveals regional measles virus transmission across West and Central Africa into Mali, while local clinical testing in Bamako shows stable sequence conservation within genotype B3.1 evolving from Nigerian sequences. The genomic information generated in this study is critical in addressing the lack of whole genome sequences available in West Africa and these findings show the importance of phylogenetically tracking measles outbreaks given recent increases in measles cases globally.

A Phylogenomic Analysis of Genipa (Rubiaceae) Using Target Sequence Capture Data

Abstract— The genus Genipa is a widespread, lowland, Neotropical lineage of trees in the coffee family, Rubiaceae. There is long-standing disagreement on the delimitation of species in the genus and how broadly Genipa is circumscribed. Here, we use genomic data to resolve the classification within Genipa. Using target sequence capture we generated a high-resolution 245-locus dataset to produce a comprehensive species phylogeny under the multi-species coalescent model. The phylogenomic results strongly support Genipa spruceana, often synonymised with Genipa americana, as a distinct monophyletic species. Similarly, the monophyly of Genipa infundibuliformis, a recently recognized species, is also strongly supported. The phylogeny also shows three distinct, well-supported clades within the widespread species, Genipa americana. These clades are interpreted as three independently evolving lineages in contrast to the two varieties most commonly recognized in G. americana based on previous morphological studies.

Phylogenomics resolves the puzzling phylogeny of banded newts (genus Ommatotriton)

Resolving the order of speciation events that occurred in rapid succession is inherently hard and typically requires a phylogenomic approach. A case in point concerns the previously unresolved phylogeny of the three species of banded newt (genus Ommatotriton). We obtain c. 7k nuclear DNA markers using target enrichment by sequence capture and analyze the dataset using maximum likelihood inference of concatenated data with RAxML, summary multi-species coalescent analysis with ASTRAL and Bayesian species tree inference using a diffusion model with SNAPPER, and use TreeMix and PhyloNet to test for interspecific gene flow. All analyses recover three distinct species with no evidence of interspecific gene flow. All analyses retrieved the topology (O. nesterovi, (O. ophryticus, O. vittatus)), with high support. SNAPPER did show the tendency to get stuck in a local optimum, resulting in a different but still highly supported topology. Furthermore, we notice that fewer SNAPPER runs get stuck in a local optimum when we include an outgroup. Therefore, we recommend the exploration of multiple independent runs and the use of an outgroup with this approach. The banded newt radiation illustrates the use of genome-wide data to tackle formerly unresolved phylogenies.

Abstract C025: Identification of cell-cell signaling programs across the tumor-tumor microenvironment ecosystem and associated with clinical status in neoadjuvant osimertinib treated patient samples revealed by spatial profiling

Abstract Lung cancer is the leading cause of cancer mortality, and despite improvements in treatment, tumors typically respond incompletely and resume growth after acquisition of drug resistance. Recent completion of a neoadjuvant osimertinib Phase II trial treating patients with surgically resectable stage I-IIIA EGFR-mutated non-small cell lung cancer (EGFRm NSCLC) (NCT03433469) (Blakely et. Al, JCO 2024) has highlighted the importance of further identifying non-genomic mechanisms of persistence and resistance to targeted therapy. Here we analyzed these patient samples to identify transcriptionally regulated signaling patterns enriched at Residual Disease (RD) after neoadjuvant osimertinib treatment compared to treatment naïve samples (TN), as well as signaling patterns enriched at RD in patients who ultimately develop disease recurrence after surgical resection compared to those who remain disease-free. Spatially resolved transcriptomic sequencing was performed, using the 10X Genomics Visium platform, on 36 tissue sections (n=9 TN, n=19 RD, n=4 Progressive Disease (PD), n=4 Tumor Adjacent Normal), from 25 patients. TN and PD samples were from standard-of-care surgical resections. 18 of 19 RD samples were from NCT03433469. The Visium array spots that tile the sequencing capture area are 55 µm in diameter, and thus can contain an estimated 1-10 cells within each array spot. After quality control, 91,582 array spots remained for downstream analysis. We identified high confidence tumor array spots annotated both as “Cancer” by a board-certified pathologist and with tumor characteristic copy number variations. Through dimensionality reduction clustering, we identified 10 major array spot clusters, including the high confidence tumor array spot cluster. These clusters were further subset into 44 high resolution clusters of array spot niches, 32 of which were shared across at least nine patients. Because array spots are not single-cell resolution, these niches represent spots with similar mixed cell type composition and transcriptional signatures. We identified significant increases in the CCL14+ Endothelial Cell Niche, CXCL2+ Endothelial Cell Niche, Lymphocytes + Endothelial Niche, and CXCL12+ Fibroblast Niche in RD samples compared to TN. We identified a cell signaling program defined by spatially co-expressed ligand-receptor pairs associated with focal adhesion, enriched in PD samples. This same pattern was enriched in RD samples from patients who had recurrent disease after surgical resection compared to RD samples from patients who remained disease free. These data describe an increase in immune trafficking in RD samples, as well as identify an EGFR- alternative signaling pathway correlating with osimertinib resistance. Identification of spatially resolved, differential architectural organization and signaling patterns in RD and PD states will expand our understanding of targeted therapy resistance in EGFRm NSCLC, with the potential to identify additional clinically actionable targets. Citation Format: Whitney Tamaki, Daniel L. Kerr, Wei Wu, Grant Eilers, Anatoly Urisman, Yu-Ting Chou, Philippe Gui, Shigeki Nanjo, Johannes R. Kratz, David M. Jablons, Trever G. Bivona, Collin M. Blakely. Identification of cell-cell signaling programs across the tumor-tumor microenvironment ecosystem and associated with clinical status in neoadjuvant osimertinib treated patient samples revealed by spatial profiling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C025.