Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo- EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Angstrom resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Angstrom. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.

Mass Spectrometry Imaging Disclosed Spatial Distribution of Defense-Related Metabolites in Triticum spp.

Fusarium Head Blight is the most common fungal disease that strongly affects Triticum spp., reducing crop yield and leading to the accumulation of toxic metabolites. Several studies have investigated the plant metabolic response to counteract mycotoxins accumulation. However, information on the precise location where the defense mechanism is taking place is scarce. Therefore, this study aimed to investigate the specific tissue distribution of defense metabolites in two Triticum species and use this information to postulate on the metabolites’ functional role, unlocking the “location-to-function” paradigm. To address this challenge, transversal cross-sections were obtained from the middle of the grains. They were analyzed using an atmospheric-pressure (AP) SMALDI MSI source (AP-SMALDI5 AF, TransMIT GmbH, Giessen, Germany) coupled to a Q Exactive HF (Thermo Fisher Scientific GmbH, Bremen, Germany) orbital trapping mass spectrometer. Our result revealed the capability of (AP)-SMALDI MSI instrumentation to finely investigate the spatial distribution of wheat defense metabolites, such as hydroxycinnamic acid amides, oxylipins, linoleic and α-linoleic acids, galactolipids, and glycerolipids.

Evaluation of Three Automated Extraction Systems for the Detection of SARS-CoV-2 from Clinical Respiratory Specimens

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly contagious and causes coronavirus disease 2019 (COVID-19). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the most accurate and reliable molecular assay to detect active SARS-CoV-2 infection. However, a rapid increase in test subjects has created a global bottleneck in testing capacity. Given that efficient nucleic acid extraction greatly affects reliable and accurate testing results, we compared three extraction platforms: MagNA Pure 96 DNA and Viral NA Small Volume kit on MagNA Pure 96 (Roche, Basel, Switzerland), careGENETM Viral/Pathogen HiFi Nucleic Acid Isolation kit (WELLS BIO Inc., Seoul, Korea) on KingFisher Flex (Thermo Fisher Scientific, Rocklin, CA, USA), and SGRespiTM Pure kit (Seegene Inc., Seoul, Korea) on Maelstrom 9600 (Taiwan Advanced Nanotech Inc., Taoyuan, Taiwan). RNA was extracted from 245 residual respiratory specimens from the different types of samples (i.e., NPS, sputum, and saliva) using three different kits. The 95% limits of detection of median tissue culture infectious dose per milliliter (TCID50/mL) for the MagNA Pure 96, KingFisher Flex, and Maelstrom 9600 were 0.37–3.15 × 101, 0.41–3.62 × 101, and 0.33–1.98 × 101, respectively. The KingFisher Flex platform exhibited 99.2% sensitivity and 100% specificity, whereas Maelstrom 9600 exhibited 98.3–100% sensitivity and 100% specificity. Bland–Altman analysis revealed a 95.2% concordance between MagNA Pure 96 and KingFisher Flex and 95.4% concordance between MagNA Pure 96 and Maelstrom 9600, indicating that all three platforms provided statistically reliable results. This suggests that two modifying platforms, KingFisher Flex and Maelstrom 9600, are accurate and scalable extraction platforms for large-scale SARS-CoV-2 clinical detection and could help the management of COVID-19 patients.

BRCA1/2 NGS Somatic Testing in Clinical Practice: A Short Report

High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of all ovarian carcinomas. HGSOC harboring BRCA1/2 germline or somatic mutations are sensitive to the poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Therefore, detecting these mutations is crucial to identifying patients for PARPi-targeted treatment. In the clinical setting, next generation sequencing (NGS) has proven to be a reliable diagnostic approach BRCA1/2 molecular evaluation. Here, we review the results of our BRCA1/2 NGS analysis obtained in a year and a half of diagnostic routine practice. BRCA1/2 molecular NGS records of HGSOC patients were retrieved from our institutional archive covering the period from January 2020 to September 2021. NGS analysis was performed on the Ion S5™ System (Thermo Fisher Scientific, Waltham, MA, USA) with the Oncomine™ BRCA Research Assay panel (Thermo Fisher Scientific). Variants were classified as pathogenic or likely pathogenic according to the guidelines of the American College of Medical Genetics and Genomics by using the inspection of Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) and ClinVar (NCBI) databases. Sixty-five HGSOC patient samples were successfully analyzed. Overall, 11 (16.9%) out of 65 cases harbored a pathogenic alteration in BRCA1/2, in particular, six BRCA1 and five BRCA2 pathogenic variations. This study confirms the efficiency and high sensitivity of NGS analysis in detecting BRCA1/2 germline or somatic variations in patients with HGSOC.

Significance of food allergy in atopic dermatitis in children

Background. Atopic dermatitis is the most common inflammatory skin disease in childhood, affecting almost 20 % of children. Food allergies affect one of three children with atopic dermatitis, and allergies to peanuts, eggs and milk are the predominant food allergies in the world. The purpose was to assess the prevalence of food allergies in the group of patients with atopic dermatitis, to determine the association of food allergies with anamnestic and clinical parameters of atopic dermatitis. Materials and me­thods. Children with atopic dermatitis (n = 116) were included in the study from September 2020 to August 2021 in Kyiv, Ukraine. Food sensitization was established by determining specific immunoglo­bulin E (sIgE) to food allergens using immunochemiluminescent method on the ImmunoCAP 100 system (Thermo Fisher Scientific Inc., Phadia, Sweden). Results. The average age of children in the main group (59 boys and 57 girls) was 6.8 years. All 116 patients at the time of examination had manifestations of atopic dermatitis in the form of erythematous itchy rash. The number of children with food allergy was 23 (19.8 %). Among food allergens, positive sIgE were found: in 14 patients (12 %) — to milk, in 13 (11.2 %) — to eggs, in 5 (4.3 %) — to fish, and in 4 people (3.4 %) — to hazelnuts. Food sensitization was significantly more common in children with moderate-to-severe atopic dermatitis than in the group of patients with mild atopic dermatitis (χ2 = 7.555, p < 0.05). Children with food sensitization had an earlier onset of atopic dermatitis — the average age of manifestations onset was 8 months, and in the group of children without food allergy, it was 18 months. Children with a parental history of atopic diseases were significantly more likely to have concomitant food allergy (χ2 = 12.831, p < 0.05). Conclusions. Given the significant association between early onset as well as moderate-to-severe atopic dermatitis and food sensitization, it is likely that food sensitization occurs primarily through an inflamed skin barrier in eczematous skin, which could potentially lead to clinical food allergy.

SARS-CoV-2 testing in the community: Testing positive samples with the TaqMan SARS-CoV-2 Mutation Panel to find variants in real-time

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages, however there can be limitations due to sequence drop-out when used to identify specific key mutations. Recently, Thermo Fisher Scientific have developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the Thermo Fisher TaqMan Mutation Panel Genotyping Assay, initially for three mutations of concern and then an additional two mutations of concern, against SARS-CoV-2 positive clinical samples and the corresponding COG-UK sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, an excellent complement to genome sequencing and has real clinical health value potential allowing laboratories to report and action variants of concern much quicker.

From Forensics to Clinical Research: Expanding the Variant Calling Pipeline for the Precision ID mtDNA Whole Genome Panel

Despite a multitude of methods for the sample preparation, sequencing, and data analysis of mitochondrial DNA (mtDNA), the demand for innovation remains, particularly in comparison with nuclear DNA (nDNA) research. The Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, USA) is an innovative library preparation kit suitable for degraded samples and low DNA input. However, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), with a heteroplasmy threshold level of 10%. Here, we present an alternative customizable pipeline, the PrecisionCallerPipeline (PCP), for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, we achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. We further validated our findings with 50 samples from an ongoing independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels of variants found with both pipelines.

Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

Introduction B cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction. Methods The B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software. Results Clonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively. Conclusions These results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders. For research use only. Disclosures Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

Evaluation of a Rapid Automated Next Generation Sequencing Assay for Precision Medicine in Acute Myeloid Leukemia

Background: In the last decade there have been significant advances in diagnosing and classifying adult acute myeloid leukemia (AML) based on genomic profiling, enabling risk-stratification and targeted therapies. In 2017 the US FDA approved the first gene mutation targeted therapies for AML with multiple additional targeted therapies since approved or in development. Given the typical acuity of AML at initial presentation however and the current turnaround time for next-generation sequencing (NGS) assays, most patients will start definitive initial therapy before all potentially targetable mutations are known. There is, therefore, a significant need for a fast molecular genotyping test to determine eligibility for personalized therapy in AML. The NCI Myeloid Assay (NMA) is a comprehensive targeted NGS assay on the Ion Torrent Genexus System, a fully automated platform that provides a rapid turnaround time from specimen receipt to clinical reporting. NMA utilizes Thermo Fisher Scientific's Oncomine Myeloid Assay GX and appears ideally suited for use in upcoming AML targeted therapy trials but has yet to be extensively tested in a cohort of AML patient diagnostic samples and compared to a standard targeted "myeloid panel" NGS assay platform (s-NGS). Methods: DNA samples (n=173) extracted from pretreatment bone marrow and/or peripheral blood of adult patients (n=112) diagnosed with de novo AML or high-risk myelodysplastic syndrome (MDS), were blindly tested in parallel using the NMA and s-NGS assays. For the NMA assay, 27.75ng of DNA was put into the Genexus System. All runs, controls, and samples were first analyzed for sequencing quality using established quality control (QC) metrics to assess pass/fail status. For all samples that passed QC metrics, variant results generated by the Ion Torrent Genexus pipeline were manually reviewed prior to being called true positive variants. For the s-NGS, using the ArcherDx Myeloid VariantPlex assay, a DNA input of 50ng was used for library preparation on a dual pre- and post-PCR separated automated liquid-handling workflow. Resulting libraries were sequencing on the Novaseq 6000 (Illumina) and the data analyzed using the Archer Analysis software and filtered as previously described (PMID: 34258102). Results from the two assays were compared for mutations with a variant allele fraction (VAF) &gt;5% occurring in genes of interest in small molecule targeted clinical trials including: FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, KRAS, and TP53. For FLT3-ITD comparison, the presence or absence of a call by the assay was used. Results: Utilizing a 5% VAF reporting threshold, a total of 171 and 174 variants were detected by NMA and s-NGS assays, respectively. A high rate of concordance was observed between the assays, with NMA detecting 96% of s-NGS variants and s-NGS detecting 95% of NMA variants. The VAF of detected single nucleotide variants was highly correlated (r=0.9848, P&lt;0.0001, Figure 1A). NPM1 mutation VAF values trended lower by s-NGS compared to NMA. We investigated the discordant calls (n=15 total in 11 patients). One patient was correctly identified as having an NRAS p.Gly12 mutation by both approaches, but the resulting mutation was incorrectly annotated by the s-NGS pipeline. Samples from two patients (including one with both blood and marrow tested) were correctly identified as being FLT3 tyrosine kinase domain mutated by both sequencing approaches, although only the major of two missense variants identified by s-NGS was reported by the NMA pipeline. None of these patients, however, would be misclassified. The remaining 11 discordant calls were false negatives (including 6 variants detected by s-NGS but not by NMA). All of these "edge case" variants were detectable by lowering the VAF reporting threshold below 5% (Figure 1B). Conclusions: NMA is an automated sample-to-results workflow that can identify myeloid disorder-associated genomic variants in less than 48 hours from library preparation to clinical reporting. We show that NMA is highly concordant with a standard DNA NGS assay for detecting mutations within recurrently mutated AML genes. Accurate rapid genotyping is required for assignment to initial treatment with targeted therapy, and this technology may be a valuable tool for upcoming clinical trials for patients with myeloid malignancies. Figure 1 Figure 1. Disclosures Zhang: Thermo Fisher Scientific: Current Employment. Sedova: Thermo Fisher Scientific: Current Employment. Huang: Thermo Fisher Scientific: Current Employment. Mittal: Thermo Fisher Scientific: Current Employment. Hatch: Thermo Fisher Scientific: Current Employment. Ni: Thermo Fisher Scientific: Current Employment. Kaznadzey: Thermo Fisher Scientific: Current Employment. Sadis: Thermo Fisher Scientific: Current Employment. Smith: Thermo Fisher Scientific: Current Employment. Williams: Illumina: Other: CRADA. Hourigan: Sellas: Research Funding.

Fully Automated Workflows Quantify and Report Key T-Cell and B-Cell Receptor Biomarkers Relevant to Immuno-Oncology and Heme-Oncology Research

Background T-cell and B-cell repertoire analysis is used in oncology research, to understand the etiology of complex disease phenotypes, for the identification of biomarkers predictive of disease burden, outcome, and response to treatment, and for research in diagnosis and recurrence monitoring. Key predictors include secondary and tertiary repertoire features not reported by existing sequencing software solutions. For example, due to ongoing somatic hypermutation in mature B-cell receptors, the underlying sequence of a given clone can accumulate base differences and appear as several distinct clones with smaller frequencies, thereby hampering the ability of analysis software to detect its presence as a single dominant clone with the highest frequency. This has particularly detrimental implications for research in disorders such as follicular lymphoma and may require clonal lineage analysis for proper mitigation. Therefore, to aid the downstream analytics of biomarker identification and the study of complex disease, we developed fully automated analysis solutions that directly compute and report several key features (clonal lineage, amongst several others described below) pertinent to this area of research. Results We developed the Oncomine™ TCR Beta-SR, TCR Gamma-SR, BCR IGH-SR and BCR IGKL-SR workflows on Ion Reporter™ to characterize T-cell (β, γ chains) and B-cell (heavy and light (κ, δ) chains) repertoires. These workflows generate output tables and visualizations for primary repertoire features such as detected clones (viz., unique rearrangements in the receptor DNA sequence), their frequencies, as well as their somatic hypermutation levels in the case of B-cells (Figure 1a & 1b) for clonality assessment and rare clone detection. The software also quantifies and reports several secondary and tertiary repertoire features in a sample, such as clonal diversity, evenness of the clonal population, and B-cell lineage groupings useful in identifying related sub-clones. It includes spectratyping format plots to simultaneously assess the above features as a function of v-gene usage and CDR3 length combinations (Figure 1c & 1d), thereby providing users a complete snapshot of the repertoire, and also the capability to quickly determine CDR3 lengths and V-gene usage of highly expanded or mutated clones. A separate CDR3 lengths histogram is included, as well as a heatmap that depicts the distributions/intensity of Variable-Joining gene combinations (Figure 1e & 1f). Furthermore, the TCR workflows also report (i) convergence frequencies (fraction of clones with different nucleotide sequences, but identical amino acid sequences), and (ii) haplotype grouping for an analyzed sample, based on V-gene allele genotyping and clustering (Figure 1g). In addition, the long read Oncomine™ BCR IGH-LR workflow uniquely reports the isotype class for every detected clone, and includes a visualization of total reads, clones and lineages in the sample represented by isotype (Figure 1h). Conclusion The Oncomine™ immune repertoire workflows for T-cell and B-cell receptor sequencing were designed to be of high utility in distinct areas of malignancy research, and we expect them to greatly simplify complex downstream analyses. The unique capabilities of the workflows to automatically report secondary and tertiary repertoire features such as (i) clonal lineages for improved dominant clone detection in blood cancers, (ii) TCR clone convergence for prediction of response to immune checkpoint inhibitors [1,2], (iii) TCR haplotype grouping for evaluation of risk factors for autoimmunity and immune-related adverse events [3], and (iv) isotype classification in BCRs for studying pan-cancer immune evasion mechanisms, demonstrate the clear advantages of using these automated workflows over other existing solutions. For research use only. References 1) Looney TJ et al. (2020) TCR Convergence in Individuals Treated With Immune Checkpoint Inhibition for Cancer. Front. Immunol. 10:2985. 2) Naidus et al. (2021) Early changes in the circulating T cells are associated with clinical outcomes after PD-L1 blockade by durvalumab in advanced NSCLC patients. Cancer Immunology, Immunotherapy 70:2095-2102 3) Looney TJ et al. (2019) Haplotype Analysis of the T-Cell Receptor Beta (TCRB) Locus by Long-amplicon TCRB Repertoire Sequencing. Journal of Immunotherapy and Precision Oncology. 2 (4): 137-143. Figure 1 Figure 1. Disclosures Sarda: Thermo Fisher Scientific: Current Employment. Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Looney: Thermo Fisher Scientific: Ended employment in the past 24 months; Singular Genomics: Current Employment. Hyland: Thermo Fisher Scientific: Current Employment.