Original Genome Research Articles

Abstract 2987: BamQuery: a new proteogenomic tool to explore the immunopeptidome and prioritize actionable tumor antigens

Abstract MHC class I-associated peptides (MAPs), collectively referred to as the immunopeptidome, have a pivotal role in cancer immunosurveillance. While MAPs were long thought to be solely generated by the degradation of canonical proteins, recent advances in the field of proteogenomics (genomically-informed proteomics) evidenced that ∼10% of them originate from allegedly noncoding genomic sequences. Among these sequences, endogenous retroelements (EREs) are under intense scrutiny as a possible source of actionable tumor antigens (TAs). With the increasing number of cancer-oriented immunopeptidomic and proteogenomic studies comes the need to accurately attribute an RNA expression level to each MAP identified by mass-spectrometry. Here, we introduce BamQuery (BQ), a computational tool to attribute an exhaustive RNA expression to MAPs of any genomic origin (exon, intron, UTR, intergenic) from bulk and single-cell RNA-sequencing data. By using BQ on large datasets of published MAPs identified by mass spectrometry, we show that many of them can arise from more than one genomic region. Indeed, 27% of MAPs reported as deriving from protein-coding exons (canonical MAPs) could also arise from non-canonical genomic regions, sometimes with greater probability, and 61% of non-canonical MAPs could arise from more than a single genomic origin (334 possible regions on average per non-canonical MAP; up to 35,343 for EREs). The consideration of all these origins evidenced an unsuspected high RNA expression in normal human tissues of (i) published neoantigens/TAs (mutated or not); (ii) MAPs derived from proteasomal splicing, supposedly not genomically templated, and (iii) MAPs derived from viruses. In particular, the high expression of candidate immunotherapeutic targets such as TAs highlights the relevance of BamQuery and the necessity of using it to validate such antigens before translating their usage in clinical trials. We also demonstrate that BamQuery can be used to directly identify safe and actionable TAs as well as to predict their immunogenicity through our freely accessible web portal (https://bamquery.iric.ca/search). Therefore, BQ could become an essential tool in any TA prioritization pipeline in the near future. Citation Format: Maria-Virginia Ruiz Cuevas, Marie-Pierre Hardy, Jean-David Larouche, Anca Apavaloaei, Eralda Kina, Krystel Vincent, Patrick Gendron, Jean-Philippe Laverdure, Chantal Durette, Pierre Thibault, Sebastien Lemieux, Claude Perreault, Gregory Ehx. BamQuery: a new proteogenomic tool to explore the immunopeptidome and prioritize actionable tumor antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2987.

Coevolution of reproducers and replicators at the origin of life and the conditions for the origin of genomes

There are two fundamentally distinct but inextricably linked types of biological evolutionary units, reproducers and replicators. Reproducers are cells and organelles that reproduce via various forms of division and maintain the physical continuity of compartments and their content. Replicators are genetic elements (GE), including genomes of cellular organisms and various autonomous elements, that both cooperate with reproducers and rely on the latter for replication. All known cells and organisms comprise a union between replicators and reproducers. We explore a model in which cells emerged via symbiosis between primordial "metabolic" reproducers (protocells) which evolved, on short time scales, via a primitive form of selection and random drift, and mutualist replicators. Mathematical modeling identifies the conditions, under which GE-carrying protocells can outcompete GE-less ones, taking into account that, from the earliest stages of evolution, replicators split into mutualists and parasites. Analysis of the model shows that, for the GE-containing protocells to win the competition and to be fixed in evolution, it is essential that the birth-death process of the GE is coordinated with the rate of protocell division. At the early stages of evolution, random, high-variance cell division is advantageous compared with symmetrical division because the former provides for the emergence of protocells containing only mutualists, preventing takeover by parasites. These findings illuminate the likely order of key events on the evolutionary route from protocells to cells that involved the origin of genomes, symmetrical cell division, and antiparasite defense systems.

Discovery of novel virulence mechanisms in Clostridium botulinum type A3 using genome-wide analysis

ObjectiveClostridium botulinum type A is a neurotoxin-producing, spore-forming anaerobic bacterium that causes botulism in humans. The evolutionary genomic context of this organism is not yet known to understand its molecular virulence mechanisms in the human intestinal tract. Hence, this study aimed to investigate the mechanisms underlying virulence and pathogenesis by comparing the genomic contexts across species, serotypes, and subtypes. MethodsA comparative genomic approach was used to analyze evolutionary genomic relationships, intergenomic distances, syntenic blocks, replication origins, and gene abundance with phylogenomic neighbors. ResultsType A strains have shown genomic proximity to group I strains with distinct accessory genes and vary even within subtypes. Phylogenomic data showed that type C and D strains were distantly related to a group I and group II strains. Synthetic plots indicated that orthologous genes might have evolved from Clostridial ancestry to subtype A3 strains, whereas syntonic out-paralogs might have emerged between subtypes A3 and A1 through α-events. Gene abundance analysis revealed the key roles of genes involved in biofilm formation, cell–cell communication, human diseases, and drug resistance compared to the pathogenic Clostridia. Moreover, we identified 43 unique genes in the type A3 genome, of which 29 were involved in the pathophysiological processes and other genes contributed to amino acid metabolism. The C. botulinum type A3 genome contains 14 new virulence proteins that can provide the ability to confer antibiotic resistance, virulence exertion and adherence to host cells, the host immune system, and mobility of extrachromosomal genetic elements. ConclusionThe results of our study provide insight into the understanding of new virulence mechanisms to discover new therapeutics for the treatment of human diseases caused by type A3 strains.

Approaches for Modes of Action Study of Long Non-Coding RNAs: From Single Verification to Genome-Wide Determination.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides (nt) that are not translated into known functional proteins. This broad definition covers a large collection of transcripts with diverse genomic origins, biogenesis, and modes of action. Thus, it is very important to choose appropriate research methodologies when investigating lncRNAs with biological significance. Multiple reviews to date have summarized the mechanisms of lncRNA biogenesis, their localization, their functions in gene regulation at multiple levels, and also their potential applications. However, little has been reviewed on the leading strategies for lncRNA research. Here, we generalize a basic and systemic mind map for lncRNA research and discuss the mechanisms and the application scenarios of 'up-to-date' techniques as applied to molecular function studies of lncRNAs. Taking advantage of documented lncRNA research paradigms as examples, we aim to provide an overview of the developing techniques for elucidating lncRNA interactions with genomic DNA, proteins, and other RNAs. In the end, we propose the future direction and potential technological challenges of lncRNA studies, focusing on techniques and applications.

Recent natural hybridization inElymusandCampeiostachysof Triticeae: evidence from morphological, cytological and molecular analyses

AbstractNatural hybrids in Triticeae have been frequently reported from the Qinghai–Tibet plateau, but minor variation in morphological features and homoploid hybridization have made it difficult to identify the origin and genome constitution of hybrids between Elymus and Campeiostachys. Specimens were investigated using morphology, cytogenetics and phylogenetic analyses to uncover the genome constitution and origin of ten putative natural hybrids (SH01-SH10) from the Qinghai–Tibet plateau. SH01, SH02, SH03, SH05 and SH06 (2n = 5x = 35, StStHHY) originated from Campeiostachys breviaristata (2n = 6x = 42, StStHHYY) and Elymus sibiricus (2n = 4x = 28, StStHH); SH04 and SH07 (2n = 5x = 35, StStHHY) originated from C. nutans (2n = 6x = 42, StStHHYY) and E. sibiricus; SH08 (2n = 5x = 35, StStHHY) originated from C. dahurica var. tangutorum (2n = 6x = 42, StStHHYY) and E. sibiricus; and SH09 and SH10 (2n = 4x = 28, StStHH) were the homoploid hybrids of E. sibiricus and an unknown Elymus sp. (2n = 4x = 28, StStHH). Karyotype variations in SH04 and SH07 might originate from the maternal progenitor. Frequent hybridization in the Qinghai–Tibet plateau might result from factors such as genome constitution, pollination habits, overlapping flowering stage, sympatric distribution and unique ecological conditions.

Allele detection using k-mer-based sequencing error profiles.

For genotype and haplotype inference, typically, sequencing reads aligned to a reference genome are used. The alignments identify the genomic origin of the reads and help to infer the absence or presence of sequence variants in the genome. Since long sequencing reads often come with high rates of systematic sequencing errors, single nucleotides in the reads are not always correctly aligned to the reference genome, which can thus lead to wrong conclusions about the allele carried by a sequencing read at the variant site. Thus, allele detection is not a trivial task, especially for single-nucleotide polymorphisms and indels. To learn the characteristics of sequencing errors, we introduce a method to create an error model in non-variant regions of the genome. This information is later used to distinguish sequencing errors from alternative alleles in variant regions. We show that our method, k-merald, improves allele detection accuracy leading to better genotyping performance as compared to the existing WhatsHap implementation using edit-distance-basedallele detection, with a decrease of 18% and 24% in error ratefor high-coverage Oxford Nanopore and PacBio CLR sequencing reads for sample HG002, respectively. We additionally observed a prominent improvement in genotyping performance for sequencing data with low coverage. For 3 coverage Oxford Nanopore sequencing data, the genotyping error rate reduced from 34% to 31%, corresponding to a 9% decrease. https://github.com/whatshap/whatshap.

Precision diagnostics in children.

Medical practice is transforming from a reactive to a pro-active and preventive discipline that is underpinned by precision medicine. The advances in technologies in such fields as genomics, proteomics, metabolomics, transcriptomics and artificial intelligence have resulted in a paradigm shift in our understanding of specific diseases in childhood, greatly enhanced by our ability to combine data from changes within cells to the impact of environmental and population changes. Diseases in children have been reclassified as we understand more about their genomic origin and their evolution. Genomic discoveries, additional 'omics' data and advances such as optical genome mapping have driven rapid improvements in the precision and speed of diagnoses of diseases in children and are now being incorporated into newborn screening, have improved targeted therapies in childhood and have supported the development of predictive biomarkers to assess therapeutic impact and determine prognosis in congenital and acquired diseases of childhood. New medical device technologies are facilitating data capture at a population level to support higher diagnostic accuracy and tailored therapies in children according to predicted population outcome, and digital ecosystems now tailor therapies and provide support for their specific needs. By capturing biological and environmental data as early as possible in childhood, we can understand factors that predict disease or maintain health and track changes across a more extensive longitudinal path. Data from multiple health and external sources over long-time periods starting from birth or even in the in utero environment will provide further clarity about how to sustain health and prevent or predict disease. In this respect, we will not only use data to diagnose disease, but precision diagnostics will aid the 'diagnosis of good health'. The principle of 'start early and change more' will thus underpin the value of applying a personalised medicine approach early in life.

Elucidation of genomic origin of synchronous endometrial and ovarian cancer (SEO) by genomic and microsatellite analysis.

Elucidation of clonal origin of synchronous endometrial and ovarian cancers (SEOs). We reviewed 852 patients who diagnosed endometrial and/or ovarian cancer. Forty-five (5.3%) patients were diagnosed as SEOs. We evaluated blood and tissue samples from 17 patients. We analyzed the clonal origins of 41 samples from 17 patients by gene sequencing, mismatch microsatellite instability (MSI) polymerase chain reaction assay and immunohistochemical (IHC) staining of 4 repair genes. Sixteen of 17 patients had at least 2 or more trunk mutations shared between endometrial and ovarian cancer suggesting the identical clonal origins. The shared trunk mutation are frequently found in endometrial cancer of the uterus, suggesting the uterine primary. Four out of 17 (24%) SEOs had mismatch repair (MMR) protein deficiency and MSI-high (MSI-H) states. One case was an endometrial carcinoma with local loss of MSH6 protein expression by IHC staining, and the result of MSI analysis using the whole formalin-fixed, paraffin-embedded specimen was microsatellite stable. In contrast, ovarian tissue was deficient MMR and MSI-H in the whole specimen. This indicated that MMR protein deficiency could occur during the progression of disease. Most SEOs are likely to be a single tumor with metastasis instead of double primaries, and their origin could be endometrium. In addition, SEOs have a high frequency of MMR gene abnormalities. These findings not only can support the notion of uterine primary, but also can help to expect the benefit for patients with SEOs by immuno-oncology treatment.

2073. Persistent HIV-1 Viremia Despite Intensive Antiviral Therapy Due to Non-responsive Clonal Viral Lineages

Abstract Background After initiation of antiviral therapy (ART), plasma HIV-1 RNA is usually undetectable after one month. In rare cases, viral suppression may not be achieved despite good adherence and with virus susceptible to ART. We used ultra-deep Primer ID next gen sequencing (NGS) to study the viral population and evolution of HIV-1 in two patients with persistent viremia on intensive ART. Methods We extracted viral RNA from plasma samples collected at multiple timepoints over the duration of the treatment from two patients (VEX1 and VEX2). We constructed Primer ID NGS libraries covering part of the pol gene and the env V1/V3 region and sequenced them on the Illumina MiSeq platform. We used the ‘tcs’ pipeline to construct template consensus sequences (TCS) for each region, and searched for drug resistance mutations (DRMs). The Geno2pheno pipeline was used to predict co-receptor tropisms. Results Patient VEX1 was followed for two years on ART. VEX2 restarted ART in the hospital and received directly observed therapy for nearly 6 months. Both had over 1 million viral copies/mL at the initiation of ART, and the CD4 cell counts were extremely low. Their viral loads slowly declined to approx. 10,000 copies/mL at the end of follow-up but complete viral suppression was not achieved for either patient despite appropriate ART. DRMs were not detected in either patient throughout the treatment with detection sensitivity as low as 0.1%. The sequencing results for VEX1 showed that there were both X4- and R5-tropic viruses at all timepoints and R5 viruses decayed much more slowly than X4 viruses, up to 35-fold more slowly in the initial 3 months of ART. Phylogenetic analysis revealed that the persistent R5 viruses were largely clonal while little clonality was found in the persistent X4 virus. In VEX2, all viruses were R5-tropic. There were three major distinct lineages in the viral population, and two of them completely disappeared after the initiation of ART while the other lineage persisted throughout therapy (Fig 1). The persistent lineage in VEX2 was highly clonal. Pooled maximum likelihood tree at env V1V3 region of 3 time points from patient VEX2. Sequences in red were from 2018 when we obtained the initial specimen from the patient. Sequences in blue were from Sept 2021 when the patient restarted ART. Sequences in green were from Oct 2021 when the patient were on ART for 3 weeks. The patient had 3 major viral lineages, L1, L2, and L3. From 2018 to Sept 2021, we could see the viral evolution of the three lineages (blue arrows). After 3 weeks of ART, L1 and L2 disappeared, while the L3 persisted. We could also see that L3 had several clonal populations which did not respond to ART nor evolve from 2018 to 2021. The additional sequences from Nov 2021 to Feb 2022 were identical to the sequences obtained on Oct 2021 (not shown on this figure). Conclusion Our study shows that persistent viremia on ART can come from clonal viral lineages that carry no DRMs. Clonally expanded and infected host cells might contribute to the phenomenon. Further study is needed to explore the origin of these clonal viral genomes. Disclosures Jonathan Parr, MD, Abbott Laboratories: Donation of laboratory testing and reagents|Gilead Sciences: Grant/Research Support|Virology Education: Honoraria|World Health Organization: Advisor/Consultant|World Health Organization: Grant/Research Support David A. Wohl, M.D., Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Janssen: Advisor/Consultant|Lilly: Grant/Research Support|Merck: Grant/Research Support|ViiV: Advisor/Consultant|ViiV: Grant/Research Support Joseph J. Eron, MD, Adagio Therapeutics: data safety monitoring committee|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Glaxo Smith Kline: Advisor/Consultant|Merck: Advisor/Consultant|ViiV Healthcare: Advisor/Consultant|ViiV Healthcare: Grant/Research Support.

Purification, full-length sequencing and genomic origin mapping of eccDNA.

Extrachromosomal circular DNA (eccDNA) was discovered more than half a century ago. However, its biogenesis and function have just begun to be elucidated. One hurdle that has prevented our understanding of eccDNA is the difficulty in obtaining pure eccDNA from cells. The current eccDNA purification methods mainly rely on depleting linear DNAs by exonuclease digestion after obtaining crude circles by alkaline lysis. Owing to eccDNA's low abundance and heterogeneous size, the current purification methods are not efficient in obtaining pure eccDNA. Here we describe a new three-step eccDNA purification (3SEP) procedure that adds a step to recover circular DNA, but not linear DNA that escape from the exonuclease digestion, whereby 3SEP results in eccDNA preparations with high purity and reproducibility. Additionally, we developed a full-length eccDNA sequencing technique by combining rolling-circle amplification with Nanopore sequencing. Accordingly, we developed a full-length eccDNA caller (Flec) to call the consensus sequence of multiple tandem copies of eccDNA contained within the debranched rolling-circle amplification product and map the consensus to its genomic origin. Collectively, our protocol will facilitate eccDNA identification and characterization, and has the potential for diagnostic and clinical applications. For a well-trained molecular biologist, it takes ~1-2 d to purify eccDNAs, another 5-6 d to carry out Nanopore library preparation and sequencing, and 1-5 d for an experienced bioinformatic scientist to analyze the data.

Demographic model and biological adaptation inferred from the genome‐wide single nucleotide polymorphism data reveal tripartite origins of southernmost Chinese Huis

AbstractObjectivesThe culturally unique Sanya Hui (SYH) people are considered descendants of ancient Cham people in Central Vietnam and exhibit a scenario of complex migration and admixture history. We aimed to characterize the genetic structure, origin, and admixture history of SYH people and explore the effect of the cultural admixture from Central/South Asia and East Asia on their genetic admixture.Materials and MethodsWe sampled 94 Hui individuals from Hainan and genotyped these samples using the Infinium Global Screening Array version 2 with over 717K SNPs. We then investigated the demographic history of SYH people by combining currently available modern and ancient genome‐wide SNP data and leveraging shared allele‐ and haplotype‐based strategies.ResultsPatterns of genetic differentiation showed that SYH people were genetically different from the northern Hui but harbored a close genomic affinity with indigenous Vietnamese. SYH had a relatively distant relationship with the Cham, which confirmed the hypothesis of the documented recent historical migration from Central Vietnam and assimilation with the Hainan indigenous people. The fitted three‐way admixture models and the reconstructed demographic frameworks showed that SYH was the admixture result with primary ancestry from East Asia and mainland Southeast Asia and minor ancestry related to Central/South Asia. GLOBETTRTER and ALDER‐based admixture time estimation revealed that genetic contact with Central Asia occurred around 28 generations ago. Our model was consistent with the frequent cultural communication along the Southern Maritime Silk Road and extensive interaction with East Asia. Besides, analyses focused on natural‐selected signatures of SYH people revealed some candidate loci associated with metabolism and immune function.DiscussionThe SYH people are considered to be Cham refugees' descendants. A long‐standing hypothesis posits that SYH derives from different genetic and cultural origins, which hypothesizes that the genetically attested admixture history of SYH people is different from northern Hui people possessing prominent Han‐related ancestry and minor western Eurasian ancestry. We found that the genetic structure of SYH people was different from Vietnam Cham and geographically diverse Hui people. Generally, three genetically attested genealogies from Central Vietnam, East Asia, and Central/South Asia in modern SYH people supported their tripartite model of genomic origins.

T3E: a tool for characterising the epigenetic profile of transposable elements using ChIP-seq data

BackgroundDespite the advent of Chromatin Immunoprecipitation Sequencing (ChIP-seq) having revolutionised our understanding of the mammalian genome’s regulatory landscape, many challenges remain. In particular, because of their repetitive nature, the sequencing reads derived from transposable elements (TEs) pose a real bioinformatics challenge, to the point that standard analysis pipelines typically ignore reads whose genomic origin cannot be unambiguously ascertained.ResultsWe show that discarding ambiguously mapping reads may lead to a systematic underestimation of the number of reads associated with young TE families/subfamilies. We also provide evidence suggesting that the strategy of randomly permuting the location of the read mappings (or the TEs) that is often used to compute the background for enrichment calculations at TE families/subfamilies can result in both false positive and negative enrichments. To address these problems, we present the Transposable Element Enrichment Estimator (T3E), a tool that makes use of ChIP-seq data to characterise the epigenetic profile of associated TE families/subfamilies. T3E weights the number of read mappings assigned to the individual TE copies of a family/subfamily by the overall number of genomic loci to which the corresponding reads map, and this is done at the single nucleotide level. In addition, T3E computes ChIP-seq enrichment relative to a background estimated based on the distribution of the read mappings in the input control DNA.We demonstrated the capabilities of T3E on 23 different ChIP-seq libraries. T3E identified enrichments that were consistent with previous studies. Furthermore, T3E detected context-specific enrichments that are likely to pinpoint unexplored TE families/subfamilies with individual TE copies that have been frequently exapted as cis-regulatory elements during the evolution of mammalian regulatory networks.ConclusionsT3E is a novel open-source computational tool (available for use at: https://github.com/michelleapaz/T3E) that overcomes some of the pitfalls associated with the analysis of ChIP-seq data arising from the repetitive mammalian genome and provides a framework to shed light on the epigenetics of entire TE families/subfamilies.

Genetic analysis of a 66-kDa protein-encoding gene of Angiostrongylus cantonensis and Angiostrongylus malaysiensis.

The rat lungworm Angiostrongylus cantonensis is globally known to be the cause of oeosinophilic meningitis in humans. Another congener, Angiostrongylus malaysiensis, is closely related to A. cantonensis and has been described as a potential human pathogenic parasite. These 2 worms are similar in terms of life cycle, host range and morphological and genetic information. However, there are limited studies about their genetic diversity based on the 66-kDa protein-encoding gene. The objective of this study was to explore the 66-kDa protein sequence variation of A. cantonensis and A. malaysiensis collected from Thailand. Two adult and 53 third-stage larval specimens of Angiostrongylus from 4 geographic locations in Thailand were molecularly identified using the 66-kDa protein gene. The phylogenetic trees (Bayesian inference tree and maximum-likelihood tree) showed that Angiostrongylus formed a monophyletic clade with a clear separation between A. cantonensis and A. malaysiensis. The genetic distance between A. cantonensis and A. malaysiensis varies from 0.82 to 2.86%, with a total of 16 variable sites. The analysis of genetic diversity revealed 1 and 5 new haplotypes of A. cantonensis and A. malaysiensis, respectively, and showed genetic differences between the populations of A. cantonensis and A. malaysiensis. The haplotype networks of A. cantonensis and A. malaysiensis populations in Thailand are similar to those of populations in some countries, indicating the range expansion of genomic origin between populations in different areas. In conclusion, the 66-kDa protein gene was a good genetic marker for studying genetic diversity and discriminating between A. cantonensis and A. malaysiensis.

Genomic Epidemiology of Vibrio cholerae O139, Zhejiang Province, China, 1994-2018.

Cholera caused by Vibrio cholerae O139 was first reported in Bangladesh and India in 1992. To determine the genomic epidemiology and origins of O139 in China, we sequenced 104 O139 isolates collected from Zhejiang Province, China, during 1994-2018 and compared them with 57 O139 genomes from other countries in Asia. Most Zhejiang isolates fell into 3 clusters (C1-C3), which probably originated in India (C1) and Thailand (C2 and C3) during the early 1990s. Different clusters harbored different antimicrobial resistance genes and IncA/C plasmids. The integrative and conjugative elements carried by Zhejiang isolates were of a new type, differing from ICEVchInd4 and SXTMO10 by single-nucleotide polymorphisms and presence of genes. Quinolone resistance-conferring mutations S85L in parC and S83I in gyrA occurred in 71.2% of the Zhejiang isolates. The ctxB copy number differed among the 3 clusters. Our findings provided new insights for prevention and control of O139 cholera .

Characterization and Identification of Probiotic Features in Lacticaseibacillus Paracasei Using a Comparative Genomic Analysis Approach.

Lacticaseibacillus paracasei species are widely used for their health-promoting properties in food and agricultural applications. These bacteria have been isolated from various habitats such as the oral cavity, cereals, vegetables, meats, and dairy products conferring them the ability to consume different carbohydrates. Two subspecies are recognized, Lacticaseibacillus paracasei subsp. paracasei and Lacticaseibacillus paracasei subsp. tolerans according to their acid production from carbohydrates. Some strains are currently used as probiotics. In this study, we performed a comparative genomic analysis of 181 genomes of the Lacticaseibacillus paracasei species to reveal genomic differences at the subspecies level and to reveal adaptive and probiotic features, and special emphasis is given to inulin consumption. No clear distinction at the subspecies level for L. paracasei was shown using a phylogenetic tree with orthologous genes from the core-genome set. In general, a good correlation was observed between genomic distance and isolation origin, suggesting that L. paracasei strains are adapted to their natural habitat, giving rise to genetic differences at the genomic level. A low frequency of undesirable characteristics such as plasmids, prophages, antibiotic resistance genes, absence of virulence factors, and frequent bacteriocin production supports these species being good candidates for use as probiotics. Lastly, we found that the inulin gene cluster in L. paracasei strains seems to differ slightly in the presence or absence of some genes but maintains a core defined by at least three fructose-PTS proteins, one hypothetical protein, and extracellular β-fructosidase. Finally, we conclude that further work has to be done for L. paracasei subspecies classification. Improving outgroup selection criteria is a key factor for their correct subspecies assignation.

In-depth analysis of genomes and functional genomics of orchid using cutting-edge high-throughput sequencing.

High-throughput sequencing technology has been facilitated the development of new methodologies and approaches for studying the origin and evolution of plant genomes and subgenomes, population domestication, and functional genomics. Orchids have tens of thousands of members in nature. Many of them have promising application potential in the extension and conservation of the ecological chain, the horticultural use of ornamental blossoms, and the utilization of botanical medicines. However, a large-scale gene knockout mutant library and a sophisticated genetic transformation system are still lacking in the improvement of orchid germplasm resources. New gene editing tools, such as the favored CRISPR-Cas9 or some base editors, have not yet been widely applied in orchids. In addition to a large variety of orchid cultivars, the high-precision, high-throughput genome sequencing technology is also required for the mining of trait-related functional genes. Nowadays, the focus of orchid genomics research has been directed to the origin and classification of species, genome evolution and deletion, gene duplication and chromosomal polyploidy, and flower morphogenesis-related regulation. Here, the progressing achieved in orchid molecular biology and genomics over the past few decades have been discussed, including the evolution of genome size and polyploidization. The frequent incorporation of LTR retrotransposons play important role in the expansion and structural variation of the orchid genome. The large-scale gene duplication event of the nuclear genome generated plenty of recently tandem duplicated genes, which drove the evolution and functional divergency of new genes. The evolution and loss of the plastid genome, which mostly affected genes related to photosynthesis and autotrophy, demonstrated that orchids have experienced more separate transitions to heterotrophy than any other terrestrial plant. Moreover, large-scale resequencing provide useful SNP markers for constructing genetic maps, which will facilitate the breeding of novel orchid varieties. The significance of high-throughput sequencing and gene editing technologies in the identification and molecular breeding of the trait-related genes in orchids provides us with a representative trait-improving gene as well as some mechanisms worthy of further investigation. In addition, gene editing has promise for the improvement of orchid genetic transformation and the investigation of gene function. This knowledge may provide a scientific reference and theoretical basis for orchid genome studies.

EP16.03-044 Genomic Evidence Depicting Clonal Evolution of Lung Adenosquamous Carcinoma

Adenosquamous carcinoma (ASC) is a rare subtype of non-small-cell lung cancer containing both adenocarcinoma (AC) and squamous cell carcinoma (SCC) components. However, the genomic background, tumor origins, and mechanisms of ASC are not fully understood.

Chromosome characterization and physical mapping of 18S rDNA in Lilium longiflorum originated interspecific hybrids using combined genomic and fluorescent in situ hybridization

This study was aimed to differentiate parental genomes, examining intergenomic composition, and mapping mitotic metaphase chromosomes by localizing parental and 18S rDNA probes in seven interspecific hybrid progenies that originated from Lilium longiflorum. Since single method of in situ hybridization has been widely used in interspecific lily breeding, while in this study, flow cytometry was used in conjunction with genomic and fluorescent in situ hybridization to determine the genomic contribution of each parent to the interspecific progenies. A significant variation was observed in the DNA content, chromosome length, and 18S loci in F1 as compared to the female and male parents. L. longiflorum showed nearly two times higher DNA content than the male parents and L. longiflorum × Asiatic progenies, but eight times higher than L. longiflorum × L. hansonii. Genomic in situ hybridization results revealed that both female and male parents contributed an equal number of chromosomes to their interspecific F1 offspring. Fluorescent in situ hybridization mapping revealed that 18S rDNA had 8, 6 and 7 loci in L. longiflorum parents, i.e., White heaven, Bright tower, and White tower, respectively, whereas each Asiatic cultivar and L. hansonii used as male showed 8 and 12 loci respectively. Interspecific progenies showed 8 and 7 loci in LA, and 10–11 in LM hybrids. These cytogenetic results implied equal genetic and chromosomal contribution from both parents to their intergenomic progenies. Therefore, this combined cytogenetic method has the potential to be genome specific and time-saving approach in lily breeding that could determine the status of hybrids and their genomic origin while achieving physical mapping and detecting genes in different genomes.

Insights from the genomes of 4 diploid Camelina spp.

Plant evolution has been a complex process involving hybridization and polyploidization making understanding the origin and evolution of a plant's genome challenging even once a published genome is available. The oilseed crop, Camelina sativa (Brassicaceae), has a fully sequenced allohexaploid genome with 3 unknown ancestors. To better understand which extant species best represent the ancestral genomes that contributed to C. sativa's formation, we sequenced and assembled chromosome level draft genomes for 4 diploid members of Camelina: C. neglecta C. hispida var. hispida, C. hispida var. grandiflora, and C. laxa using long and short read data scaffolded with proximity data. We then conducted phylogenetic analyses on regions of synteny and on genes described for Arabidopsis thaliana, from across each nuclear genome and the chloroplasts to examine evolutionary relationships within Camelina and Camelineae. We conclude that C. neglecta is closely related to C. sativa's sub-genome 1 and that C. hispida var. hispida and C. hispida var. grandiflora are most closely related to C. sativa's sub-genome 3. Further, the abundance and density of transposable elements, specifically Helitrons, suggest that the progenitor genome that contributed C. sativa's sub-genome 3 maybe more similar to the genome of C. hispida var. hispida than that of C. hispida var. grandiflora. These diploid genomes show few structural differences when compared to C. sativa's genome indicating little change to chromosome structure following allopolyploidization. This work also indicates that C. neglecta and C. hispida are important resources for understanding the genetics of C. sativa and potential resources for crop improvement.

Genome origin and evolution of common oat.

Genome origin and evolution of common oat.