DNA Sequencing Approach Research Articles

Tracking Response and Resistance in Acute Myeloid Leukemia through Single-Cell DNA Sequencing Helps Uncover New Therapeutic Targets.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasia with a complex polyclonal architecture. Among driver lesions, those involving the FLT3 gene represent the most frequent mutations identified at diagnosis. The development of tyrosine kinase inhibitors (TKIs) has improved the clinical outcomes of FLT3-mutated patients (Pt). However, overcoming resistance to these drugs remains a challenge. To unravel the molecular mechanisms underlying therapy resistance and clonal selection, we conducted a longitudinal analysis using a single-cell DNA sequencing approach (MissionBioTapestri® platform, San Francisco, CA, USA) in two patients with FLT3-mutated AML. To this end, samples were collected at the time of diagnosis, during TKI therapy, and at relapse or complete remission. For Pt #1, disease resistance was associated with clonal expansion of minor clones, and 2nd line TKI therapy with gilteritinib provided a proliferative advantage to the clones carrying NRAS and KIT mutations, thereby responsible for relapse. In Pt #2, clonal architecture was less complex, and 1st line TKI therapy with midostaurin was able to eradicate the leukemic clones. Our results corroborate previous findings about clonal selection driven by TKIs, highlighting the importance of a deeper characterization of individual clonal architectures for choosing the best treatment plan for personalized approaches aimed at optimizing outcomes.

A deep-sea isopod that consumes Sargassum sinking from the ocean's surface.

Most deep-ocean life relies on organic carbon from the surface ocean. While settling primary production rapidly attenuates in the water column, pulses of organic material can be quickly transported to depth in the form of food falls. One example of fresh material that can reach great depths across the tropical Atlantic Ocean and Caribbean Sea is the pelagic macroalgae Sargassum. However, little is known about the deep-ocean organisms able to use this food source. Here, we encountered the isopod Bathyopsurus nybelini at depths 5002-6288 m in the Puerto Rico Trench and Mid-Cayman Spreading Center using the Deep Submergence Vehicle Alvin. In most of the 32 observations, the isopods carried fronds of Sargassum. Through an integrative suite of morphological, DNA sequencing, and microbiological approaches, we show that this species is adapted to feed on Sargassum by using a specialized swimming stroke, having serrated and grinding mouthparts, and containing a gut microbiome that provides a dietary contribution through the degradation of macroalgal polysaccharides and fixing nitrogen. The isopod's physiological, morphological, and ecological adaptations demonstrate that vertical deposition of Sargassum is a direct trophic link between the surface and deep ocean and that some deep-sea organisms are poised to use this material.

Seasonal variation in the stomach microbiota of two sympatrically breeding Pygoscelis penguin species at Signy Island, South Orkney Islands.

The gut microbiomes of Antarctic penguins are important for the fitness of the host birds and their chicks. The compositions of microbial communities in Antarctic penguin guts are strongly associated with the birds' diet, physiological adaptation and phylogeny. Whilst seasonal changes in food resources, distribution and population parameters of Antarctic penguins have been well addressed, little research is available on the stability or variability of penguin stomach microbiomes over time. Here, we focused on two Pygoscelis penguin species breeding sympatrically in the maritime Antarctic and analysed their stomach contents to assess whether penguin gut microbiota differed over three austral summer breeding seasons. We used a high-throughput DNA sequencing approach to study bacterial diversity in stomach regurgitates of Adélie (Pygoscelis adeliae) and chinstrap (Pygoscelis antarctica) penguins that have a similar foraging regime on Signy Island (South Orkney Islands). Our data revealed significant differences in bacterial alpha and beta diversity between the study seasons. We also identified bacterial genera that were significantly associated with specific breeding seasons, diet compositions, chick-rearing stages and sampling events. This study provides a baseline for establishing future monitoring of penguin gut microbiomes in a rapidly changing environment.

Activated sludge prokaryote and eukaryote characterization in a pulp mill facility using amplicon sequencing

The pulp and paper industry, a major global sector, supports economies and jobs while contributing to various products. While providing valuable products, and despite Best Available Techniques (BAT) being used, managing wastewater effectively remains a key area for developing technologies and alternatives for environmental protection. Activated sludge (AS) systems are commonly used for effluent treatment, where microorganisms composition influences reactor efficiency. Current monitoring strategies show limited correlation with chemical parameters. Metabarcoding, a DNA sequencing approach, offers a promising alternative to profile microbial communities comprehensively. Utilizing MinION and Illumina sequencing, this study reveals the prokaryotic and eukaryotic microbial communities in AS from an extended aeration-activated sludge reactor from a pulp mill for the first time. Eukaryotic composition evaluated by 18S amplicon sequencing revealed Protists as the dominant group, followed by Animalia and Fungi. Within these groups, the most abundant phyla detected were Ciliophora (75 % on average), Rotifera (90 % on average), and Cryptomycota (60 % on average), respectively. Genera like Prorodon and Limnias stand out due to their high relative abundance within the Protist and Animalia group, respectively. A comparative analysis with routine microorganism composition assessment by microscopy revealed consistent taxonomic trends, affirming the feasibility of amplicon sequencing for routine microbial community surveillance. Furthermore, the incorporation of metabarcoding enhanced the depth of results. Prokaryotic composition analyzed by 16S amplicon sequencing revealed microorganisms previously described as relevant to the wastewater treatment process but highlighted dominant specific groups even when compared to other pulp facilities. Genera like Terrimonas, Defluviicoccus, and Methylocaldum appear as the most abundant among the identified genera. For proper reactor operation, microorganisms must aggregate around organic matter in flocs, which reflect reactor health and are composed of 5 % eukaryotes and 95 % prokaryotes. Hence, the characterization of both groups of microorganisms is crucial. Differences in communities between the wastewater treatment plant's aeration zones emphasize the importance of comprehensive community characterization. The study underscores metabarcoding's potential as a monitoring tool, for future linking of microbial communities with process patterns for improved environmental management in the pulp and paper industry and emphasizes the importance of establishing unique baselines for microbial communities in activated sludge systems.

Population genomics of the endangered freshwater mussel, Arcidens wheeleri (Unionoidea: Unionidae: Anodontini), in the Little River, Arkansas, USA

ABSTRACT North America is a hotspot of freshwater mussel diversity. However, over the last century, many anthropogenic stressors have led to substantial declines in freshwater mussels of the families Unionidae and Margaritiferidae in North America. Conserving the remaining mussel fauna is a priority, as they play an integral role in freshwater ecosystems. The Little River in Arkansas is home to one of the last remaining populations of the federally endangered freshwater mussel, Arcidens wheeleri (Unionidae: Anodontini). Population dynamics information is lacking for A. wheeleri, and no population genetics study has been conducted on this species. A greater understanding of the genetic diversity within a population can serve as a benchmark for developing an effective management plan. We sampled A. wheeleri from three locations in the Little River. Genomic data were generated with a single-enzyme restriction-site-associated DNA sequencing approach to assess genetic diversity and structure of A. wheeleri in the Little River. Genetic structure analyses indicated one genetic population among the three locations, with limited, fine-scale subpopulation structure. Observed heterozygosity values were considerably lower than expected heterozygosity values, with Ho = 0.14 and He = 0.22, likely indicating a genetic bottleneck. Demographic analysis of the Little River population of A. wheeleri also suggests a historical bottleneck. Furthermore, a high inbreeding coefficient (FIS = 0.33) indicates A. wheeleri in the Little River is losing genetic diversity. Data generated indicate considerable risk of extirpation for A. wheeleri from the Little River and should serve as a baseline for future monitoring. Given its high risk of extinction, we recommend increased study of A. wheeleri across its range and on-the-ground conservation actions that include habitat protection and restoration, which are the only options until a successful host fish and protocols are identified for propagation.

Conservation genomics assessment of Tharp's bluestar (Amsonia tharpii) with comparisons to widespread (A. longilora) and narrowly endemic (A. fugatei) congeners.

Land-use change and habitat fragmentation are threats to biodiversity. The decrease in available habitat, increase in isolation, and mating within populations can lead to elevated inbreeding, lower genetic diversity, and poor fitness. Here we investigate the genetics of two rare and threatened plant species, Amsonia tharpii and A. fugatei, and we compare them to a widespread congener A. longiflora. We also report the first phylogenetic study of the genus Amsonia (Apocynaceae), including 10 of the 17 taxa and multiple sampling locations, to understand species relationships. We used a double digest restriction-site associated DNA sequencing (ddRADseq) approach to investigate the genetic diversity and gene flow of each species and to create a maximum likelihood phylogeny. The ddRADseq data was mapped to a reference genome to separate out the chloroplast and nuclear markers for population genetic analysis. Our results show that genetic diversity and inbreeding were low across all three species. The chloroplast and nuclear dataset in A. tharpii were highly structured, whereas they showed no structure for A. fugatei, while A. longiflora lacked structure for nuclear data but not chloroplast. Phylogenetic results revealed that A. tharpii is distinct and sister to A. fugatei, and together they are distantly related to A. longiflora. Our results demonstrated that evolutionary history and contemporary ecological processes largely influences genetic diversity within Amsonia. Interestingly, we show that in A. tharpii there was significant structure despite being pollinated by large, bodied hawkmoths that are known to be able to carry pollen long distances, suggesting that other factors are contributing to the structure observed among A. tharpii populations. Conservation efforts should focus on protecting all of the A. tharpii populations, as they contain unique genetic diversity, and a protection plan for A. fugatei needs to be established due to its limited distribution.

Adhesive and biofilm-forming Candida glabrata Lebanese hospital isolates harbour mutations in subtelomeric silencers and adhesins.

The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1.

Fine-scale adaptive divergence and population genetic structure of Aedes aegypti in Metropolitan Manila, Philippines

BackgroundThe adaptive divergence of Aedes aegypti populations to heterogeneous environments can be a driving force behind the recent expansion of their habitat distribution and outbreaks of dengue disease in urbanized areas. In this study, we investigated the population genomics of Ae. aegypti at a regional scale in Metropolitan Manila, Philippines.MethodsWe used the Pool-Seq double digestion restriction-site association DNA sequencing (ddRAD-Seq) approach to generate a high number of single nucleotide polymorphisms (SNPs), with the aim to determine local adaptation and compare the population structure with 11 microsatellite markers. A total of 217 Ae. aegypti individuals from seven female and seven male populations collected from Metropolitan Manila were used in the assays.ResultsWe detected 65,473 SNPs across the populations, of which 76 were non-neutral SNPs. Of these non-neutral SNPs, the multivariate regression test associated 50 with eight landscape variables (e.g. open space, forest, etc.) and 29 with five climate variables (e.g. air temperature, humidity, etc.) (P-value range 0.005–0.045) in female and male populations separately. Male and female populations exhibited contrasting spatial divergence, with males exhibiting greater divergence than females, most likely reflecting the different dispersal abilities of male and female mosquitoes. In the comparative analysis of the same Ae. aegypti individuals, the pairwise FST values of 11 microsatellite markers were lower than those of the neutral SNPs, indicating that the neutral SNPs generated via pool ddRAD-Seq were more sensitive in terms of detecting genetic differences between populations at fine-spatial scales.ConclusionsOverall, our study demonstrates the utility of pool ddRAD-Seq for examining genetic differences in Ae. aegypti populations in areas at fine-spatial scales that could inform vector control programs such as Wolbachia-infected mosquito mass-release programs. This in turn would provide information on mosquito population dispersal patterns and the potential barriers to mosquito movement within and around the release area. In addition, the potential of environmental adaptability observed in Ae. aegypti could help population control efforts.Graphical

Double‐digest restriction‐associated DNA sequencing‐based genotyping and its applications in sesame germplasm management

AbstractSesame (Sesamum indicum L.) is an ancient oilseed crop belonging to the family Pedaliaceae and a globally cultivated crop for its use as oil and food. In this study, 2496 sesame accessions, being conserved at the National Genebank of ICAR‐National Bureau of Plant Genetic Resources (NBPGR), were genotyped using genomics‐assisted double‐digest restriction‐associated DNA sequencing (ddRAD‐seq) approach. A total of 64,910 filtered single‐nucleotide polymorphisms (SNPs) were utilized to assess the genome‐scale diversity. Applications of this genome‐scale information (reduced representation using restriction enzymes) are demonstrated through the development of a molecular core collection (CC) representing maximal SNP diversity. This information is also applied in developing a mid‐density panel (MDP) comprising 2515 hyper‐variable SNPs, representing almost equally the genic and non‐genic regions. The sesame CC comprising 384 accessions, a representative set of accessions with maximal diversity, was identified using multiple criteria such as k‐mer (subsequence of length “k” in a sequence read) diversity, observed heterozygosity, CoreHunter3, GenoCore, and genetic differentiation. The coreset constituted around 15% of the total accessions studied, and this small subset had captured >60% SNP diversity of the entire population. In the coreset, the admixture analysis shows reduced genetic complexity, increased nucleotide diversity (π), and is geographically distributed without any repetitiveness in the CC germplasm. Within the CC, India‐originated accessions exhibit higher diversity (as expected based on the center of diversity concept), than those accessions that were procured from various other countries. The identified CC set and the MDP will be a valuable resource for genomics‐assisted accelerated sesame improvement program.

Weighted single step GWAS reveals genomic regions associated with economic traits in Murrah buffaloes

The objective of the present study was to identify genomic regions influencing economic traits in Murrah buffaloes using weighted single step Genome Wide Association Analysis (WssGWAS). Data on 2000 animals, out of which 120 were genotyped using a double digest Restriction site Associated DNA (ddRAD) sequencing approach. The phenotypic data were collected from NDRI, India, on growth traits, viz., body weight at 6M (month), 12M, 18M and 24M, production traits like 305D (day) milk yield, lactation length (LL) and dry period (DP) and reproduction traits like age at first calving (AFC), calving interval (CI) and first service period (FSP). The biallelic genotypic data consisted of 49353 markers post-quality check. The heritability estimates were moderate to high, low to moderate, low for growth, production, reproduction traits, respectively. Important genomic regions explaining more than 0.5% of the total additive genetic variance explained by 30 adjacent SNPs were selected for further analysis of candidate genes. In this study, 105 genomic regions were associated with growth, 35 genomic regions with production and 42 window regions with reproduction traits. Different candidate genes were identified in these genomic regions, of which important are OSBPL8, NAP1L1 for growth, CNTNAP2 for production and ILDR2, TADA1 and POGK for reproduction traits.

Assessing the Plastisphere from Floating Plastics in the Northwestern Mediterranean Sea, with Emphasis on Viruses.

Plastics in the ocean create the "plastisphere", a diverse habitat hosting various life forms. Other than the pollution induced by plastics, the co-occurrence of primary producers, symbiotic organisms, decomposers, and pathogens within the plastisphere raises questions about how they influence the dynamics of marine ecosystems. Here, we used a shotgun DNA-sequencing approach to describe the species thriving on floating plastics collected in two Mediterranean sites. Our findings revealed many species of bacteria, eukaryotes, viruses, and archaea on each plastic. Proteobacteria was dominant (70% of reads in the entire dataset), with other groups such as Ascomycota fungi (11%) and Bacteroidetes (9%) also being represented. The community structure was not affected by the polymeric composition or the plastic shape. Notably, pathogenic Vibrio species, including V. campbelli, V. alginolyticus, and V. coralliilyticus, were among the most abundant species. Viruses, despite showing lower relative abundances, occurred in all samples, especially Herpesvirales, Caudovirales, and Poxviridae groups. A significant finding was the presence of the White Spot Syndrome virus (WSSV). This pathogen, responsible for devastating outbreaks in aquaculture systems, had not been previously reported in the marine plastisphere. Our study emphasizes the need for further investigation into the ecological and economic impacts of plastisphere organisms in the ocean.

Microscopic and metatranscriptomic analyses revealed unique cross-domain parasitism between phylum Candidatus Patescibacteria/candidate phyla radiation and methanogenic archaea in anaerobic ecosystems.

To verify whether members of the phylum Candidatus Patescibacteria parasitize archaea, we applied cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses on the Patescibacteria-enriched cultures derived from a methanogenic bioreactor. Amendment of cultures with exogenous methanogenic archaea, acetate, amino acids, and nucleoside monophosphates increased the relative abundance of Ca. Patescibacteria. The predominant Ca. Patescibacteria were families Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, and the former showed positive linear relationships (r2 ≥ 0.70) Methanothrix in their relative abundances, suggesting related growth patterns. Methanothrix and Methanospirillum cells with attached Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, respectively, had significantly lower cellular activity than those of the methanogens without Ca. Patescibacteria, as extrapolated from fluorescence in situ hybridization-based fluorescence. We also observed that parasitized methanogens often had cell surface deformations. Some Methanothrix-like filamentous cells were dented where the submicron cells were attached. Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae highly expressed extracellular enzymes, and based on structural predictions, some contained peptidoglycan-binding domains with potential involvement in host cell attachment. Collectively, we propose that the interactions of Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae with methanogenic archaea are parasitisms.IMPORTANCECulture-independent DNA sequencing approaches have explored diverse yet-to-be-cultured microorganisms and have significantly expanded the tree of life in recent years. One major lineage of the domain Bacteria, Ca. Patescibacteria (also known as candidate phyla radiation), is widely distributed in natural and engineered ecosystems and has been thought to be dependent on host bacteria due to the lack of several biosynthetic pathways and small cell/genome size. Although bacteria-parasitizing or bacteria-preying Ca. Patescibacteria have been described, our recent studies revealed that some lineages can specifically interact with archaea. In this study, we provide strong evidence that the relationship is parasitic, shedding light on overlooked roles of Ca. Patescibacteria in anaerobic habitats.

KAS-seq profiling captures transcription dynamics during oocyte maturation

In fully grown oocytes, the genome is considered to be globally transcriptionally silenced. However, this conclusion is primarily derived from the results obtained through immunofluorescence staining or inferred from the highly condensed state of chromosomes, lacking more direct evidence. Here, by using a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, we investigated the landscape of single-strand DNA (ssDNA) throughout the genome and provided a readout of the activity and dynamics of transcription during oocyte meiotic maturation. In non-surrounded nucleolus (NSN) oocytes, we observed a robust KAS-seq signal, indicating the high transcriptional activity. In surrounded nucleolus (SN) oocytes, the presence of ssDNA still persists although the KAS-seq signal was relatively weak, suggesting the presence of transcription. Accompanying with the meiotic resumption, the transcriptional activity gradually decreased, and global repression was detected in matured oocytes. Moreover, we preformed the integrative genomics analysis to dissect the transcriptional dynamics during mouse oocyte maturation. In sum, the present study delineates the detailed transcriptional activity during mammalian oocyte maturation.

Genetic diversity, population structure analysis and codon substitutions of Indicine Badri cattle using ddRAD sequencing.

The present study was carried out on 96 animals representing three distinct colour variants of Badri cattle to investigate the genetic diversity, population structure and substitution mutations in the genetic codons due to single nucleotide variations. The DNA samples of 96 Badri cows were genotyped using a double digestion restriction associated DNA (ddRAD) sequencing approach. A standardized bioinformatics pipeline was employed to identify single nucleotide polymorphisms (SNPs), initially detecting 7,168,552 SNPs through alignment with the Bos indicus reference genome assembly. Subsequent stringent quality filtration yielded 65,483 high-confidence SNPs for downstream analysis. Genetic diversity analysis of the Badri cattle population resulted in average values of 0.145, 0.088, and 0.091 for Shannon's diversity Index (I), Simpson's Diversity (h), and Simpson's Unbiased Diversity (uh), respectively. Genetic similarities between the black and brown, black and grey, and brown and grey Badri variantswere found to be 0.9972, 0.9980 and 0.9970, respectively. Tajima's D diversity value was observed to be significant and positive for 99.29% of high-confidence SNPs (65,483). STRUCTURE analysis showed admixture among the three Badri colour variants, suggesting a lack of genetic differentiation. Annotation of high-confidence SNPs regarding genetic codon changes indicated maximum substitutions in the GGC with GGT (22 occurrences), followed by AAC to AGC (20 occurrences), GAA to TAA (19 occurrences) and CAA to CAG (19 occurrences). The study concludes there are genetic similarities among colour variants, lack of rare alleles, balancing selection, sudden population contraction and genetic codon substitutions within the Badri cattle population. Insights derived from SNP data analysis hold potential significance for conservation initiatives and breed improvement programs for indicine cattle.

Ecological niche modelling and population genomics provide insights into the geographic and demographic ‘explosion’ of a non‐indigenous salmonid

AbstractAimEffective management of non‐indigenous species requires knowledge of their dispersal factors and founder events. We aim to identify the main environmental drivers favouring dispersal events along the invasion gradient and to characterize the spatial patterns of genetic diversity in feral populations of the non‐native pink salmon within its epicentre of invasion in Norway.LocationMainland Norway and North Atlantic Basin.MethodsWe first conducted SDM using four modelling techniques with varying levels of complexity, which encompassed both regression‐based and tree‐based machine‐learning algorithms, using climatic data from the present to 2050. Then, we used the triple‐enzyme restriction‐site associated DNA sequencing (3RADseq) approach to genotype over 30,000 high‐quality single‐nucleotide polymorphisms to elucidate the patterns of genetic diversity and gene flow within the pink salmon putative invasion hotspot.ResultsWe discovered temperature‐ and precipitation‐related variables drove pink salmon distributional shifts across its non‐native ranges and that climate‐induced favourable areas will remain stable for the next 30 years. In addition, all SDMs identified north‐eastern Norway as the epicentre of the pink salmon invasion, and genomic data revealed that there was minimal variation in genetic diversity across the sampled populations at a genome‐wide level in this region. While utilizing a specific group of ‘diagnostic’ SNPs, we observed a significant degree of genetic differentiation, ranging from moderate to substantial, and detected four hierarchical genetic clusters concordant with geography.Main ConclusionsOur findings suggest that fluctuations in climate extreme events associated with ongoing climate change will likely maintain environmental favourability for the pink salmon outside its ‘native’/introduced ranges. Locally invaded rivers are themselves potential source populations of invaders in the ongoing secondary spread of pink salmon in Northern Norway. Our study shows that SDMs and genomic data can reveal species distribution determinants and provide indicators to aid in post‐control measures and potentially inferences about their success.

Prospective Molecular Characterization of Multiple Myeloma Patient Samples Identifies High-Risk Patients and Informs Treatment Sequences through Resistance Mechanisms to Immunotherapies

Introduction. Multiple myeloma (MM) genetic diagnostics has traditionally been performed using fluorescence in situ hybridization (FISH), giving prognostic information based on immunoglobulin (Ig) translocations and key copy number abnormalities such as 1p, 1q, and 17p. However, in recent years it has become clear that more mutations, biallelic events, and focal deletions are also prognostically important and can be captured better through molecular techniques such as DNA sequencing. In addition, as targeted and immunotherapies become standard, monitoring the molecular targets will be important in determining the optimal sequence of therapies. Methods. Bone marrow aspirates from patients with MGUS/amyloid (n=3), smoldering MM (n=3), newly diagnosed MM (n=15), or progression/relapse MM (n=33) were collected. All patients were enrolled in the Indiana Myeloma Registry and consented for use of data for research purposes. CD138+ MACS cell separation was performed in a clinical pathology laboratory and DNA was extracted from CD138+ cells and patient-matched buccal swabs. 100 ng DNA from both the tumor and control sample were processed using the HyperPlus kit (KAPA Biosystems) and hybridized to the Myeloma Genome Project (MGP) sequencing panel in a CLIA-certified laboratory. The panel consisted of 228 genes for mutation and copy number analysis, as well as the immunoglobulin and MYC loci for translocation analysis. Samples were sequenced using paired-end reads to a median depth of 1063x. Data were analyzed using a clinical workflow and genomic abnormalities reported by a pathologist. Results. Of the 54 patients, Ig translocations were detected in 30 and included the known t(4;14), t(6;14), t(11;14), t(14;16) and t(12;14) as well as rare Ig translocations to partners such as GADD45B, LYN, PPCDC, and RCBTB2, of which over-expression of GADD45B and LYN are potentially targetable. A complex t(1;8;11;14) was also detected involving CCND1, MYC and the IGH loci which would not have been detectable by FISH. High-risk markers, such as gain/amp1q (22.2%) and deletion/mutation TP53 (16.6%) were detected including biallelic TP53 alterations in 5 patients (9.3%), which were mostly deletion plus mutation (4/5) and would not have been detected by traditional techniques. Of the MM patients who had been previously treated (n=33), 27 had received an IMiD and 30 had received a proteasome inhibitor. One patient showed mutation of CRBN (p.N236D) in conjunction with del3p and another patient with mutation of CUL4B (p.E277Ter) with delX indicating complete inactivation of the genes as a resistance mechanism to IMiDs. In addition, 17 patients had received an immunotherapeutic regimen including anti-CD38 or anti-BCMA. Of those that had received an anti-CD38 treatment (n=17), three had deletion of 4p (the location of CD38) including one patient, who had received three previous anti-CD38 regimens, with focal deletion of CD38. Six patients had also received anti-BCMA therapy, of which one had a mutation in the gene encoding BCMA, TNFRSF17, resulting in premature termination within the extracellular domain (p.S48Ter) in 42% of cells. This sample came from a patient who relapsed after an initial response to a BCMA CAR-T, followed by a progression occurring quickly while on a BCMA bispecific antibody Teclistamab, suggesting the loss of BCMA expression as a mechanism of resistance in both treatments. Interestingly, two patients harbored somatic mutations in the transmembrane domains of either CD38 (p.S29I) or TNFRSF17 (p.L60S) prior to the start of the respective treatments indicating that not all mutations seen in these genes are a result of treatment resistance. Conclusions. DNA sequencing approaches are cost-effective alternatives to FISH, the current standard diagnostic technology, to detect genomic abnormalities in multiple myeloma and they can identify clinically meaningful markers related to prognosis and treatment, particularly in the emerging era of immunotherapeutics where antigen evasion is a key mechanism of molecular resistance.

Single-nucleus DNA sequencing reveals hidden somatic loss-of-heterozygosity in Cerebral Cavernous Malformations

Cerebral Cavernous Malformations (CCMs) are vascular malformations of the central nervous system which can lead to moderate to severe neurological phenotypes in patients. A majority of CCM lesions are driven by a cancer-like three-hit mutational mechanism, including a somatic, activating mutation in the oncogene PIK3CA, as well as biallelic loss-of-function mutations in a CCM gene. However, standard sequencing approaches often fail to yield a full complement of pathogenic mutations in many CCMs. We suggest this reality reflects the limited sensitivity to identify low-frequency variants and the presence of mutations undetectable with bulk short-read sequencing. Here we report a single-nucleus DNA-sequencing approach that leverages the underlying biology of CCMs to identify lesions with somatic loss-of-heterozygosity, a class of such hidden mutations. We identify an alternative genetic mechanism for CCM pathogenesis and establish a method that can be repurposed to investigate the genetic underpinning of other disorders with multiple somatic mutations.

High-volume hybridoma sequencing on the NeuroMabSeq platform enables efficient generation of recombinant monoclonal antibodies and scFvs for neuroscience research

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

Identifying Diversity of Thrips on Lablab (Lablab purpureus): A Potential Vegetable Crop

Background: Lablab (Lablab purpureus) is a potential vegetable crop cultivated throughout India. It is a tropical and subtropical vegetable adaptive to different climatic conditions and grown in all seasons. Thrips are one of the important pests of lablab as they affect all stages of the crop and transmit plant viruses. The current study aimed at identifying the thrips diversity present in lablab as a base for monitoring and management purposes. Methods: An exploratory survey was conducted to identify the diversity of thrips on lablab in its growing areas of Coimbatore. Furthermore, continuous monitoring of the lablab crops in TNAU orchard, Coimbatore, was done to explore all the life stages of thrips species and their diversity. Morphological and molecular characterization of the collected species was done using taxonomic keys and mtCOI DNA sequencing approach. Result: Thrips palmi and Thrips tabaci were observed during the crop’s early stages before blossoming. Frankliniella schultzei is a polyphagous pest species that has been found infesting all phases of crop development, from early leaf through pod formation. During and after blooming, the four species Megalurothrips usitatus, Thrips parvispinus, Chaetanaphothrips orchidii and Haplothrips gowdeyi began to infest. Shannon and Simpson diversity indices were determined to be -1.739 and -0.1979. According to Shannon index, C. orchidii and T. parvispinus were the first and second predominant species after flowering. Taxonomic investigations of the collected species were done and substantiated with molecular identification of those analyzed species. A phylogenetic tree of the analyzed sequences was constructed to denote species divergence based on their specific sequences.

Integrative omics approaches to advance rare disease diagnostics.

Over the past decade high-throughput DNA sequencing approaches, namely whole exome and whole genome sequencing became a standard procedure in Mendelian disease diagnostics. Implementation of these technologies greatly facilitated diagnostics and shifted the analysis paradigm from variant identification to prioritisation and evaluation. The diagnostic rates vary widely depending on the cohort size, heterogeneity and disease and range from around 30% to 50% leaving the majority of patients undiagnosed. Advances in omics technologies and computational analysis provide an opportunity to increase these unfavourable rates by providing evidence for disease-causing variant validation and prioritisation. This review aims to provide an overview of the current application of several omics technologies including RNA-sequencing, proteomics, metabolomics and DNA-methylation profiling for diagnostics of rare genetic diseases in general and inborn errors of metabolism in particular.