Sample Barcoding Research Articles

Polyphonia: detecting inter-sample contamination in viral genomic sequencing data.

In viral genomic research and surveillance, inter-sample contamination can affect variant detection, analysis of within-host evolution, outbreak reconstruction, and detection of superinfections and recombination events. While sample barcoding methods exist to track inter-sample contamination, they are not always used and can only detect contamination in the experimental pipeline from the point they are added. The underlying genomic information in a sample, however, carries information about inter-sample contamination occurring at any stage. Here, we present Polyphonia, a tool for detecting inter-sample contamination directly from deep sequencing data without the need for additional controls, using intrahost variant frequencies. We apply Polyphonia to 1 102 SARS-CoV-2 samples sequenced at the Broad Institute and already tracked using molecular barcoding for comparison. Polyphonia is available as a standalone Docker image and is also included as part of viral-ngs, available in Dockstore. Full documentation, source code, and instructions for use are available at https://github.com/broadinstitute/polyphonia. Data for reproducing results are available at Bioinformatics online.

High accuracy meets high throughput for near full-length 16S ribosomal RNA amplicon sequencing on the Nanopore platform.

Small subunit (SSU) ribosomal RNA (rRNA) gene amplicon sequencing is a foundational method in microbial ecology. Currently, short-read platforms are commonly employed for high-throughput applications of SSU rRNA amplicon sequencing, but at the cost of poor taxonomic classification due to limited fragment lengths. The Oxford Nanopore Technologies (ONT) platform can sequence full-length SSU rRNA genes, but its lower raw-read accuracy has so-far limited accurate taxonomic classification and de novo feature generation. Here, we present a sequencing workflow, termed ssUMI, that combines unique molecular identifier (UMI)-based error correction with newer (R10.4+) ONT chemistry and sample barcoding to enable high throughput near full-length SSU rRNA (e.g. 16S rRNA) amplicon sequencing. The ssUMI workflow generated near full-length 16S rRNA consensus sequences with 99.99% mean accuracy using a minimum subread coverage of 3×, surpassing the accuracy of Illumina short reads. The consensus sequences generated with ssUMI were used to produce error-free de novo sequence features with no false positives with two microbial community standards. In contrast, Nanopore raw reads produced erroneous de novo sequence features, indicating that UMI-based error correction is currently necessary for high-accuracy microbial profiling with R10.4+ ONT sequencing chemistries. We showcase the cost-competitive scalability of the ssUMI workflow by sequencing 87 time-series wastewater samples and 27 human gut samples, obtaining quantitative ecological insights that were missed by short-read amplicon sequencing. ssUMI, therefore, enables accurate and low-cost full-length 16S rRNA amplicon sequencing on Nanopore, improving accessibility to high-resolution microbiome science.

Report of a new species of sand fly, Phlebotomus (Anaphlebotomus) ajithii n. sp. (Diptera: Psychodidae), from Western Ghats, India

BackgroundWestern Ghats is a biodiversity treasure trove with reports of indigenous leishmaniasis cases. Hence, systematic sand fly surveillance was carried out among the tribal population. The present study reports a novel sand fly species, Phlebotomus (Anaphlebotomus) ajithii n. sp. (Diptera: Psychodidae), discovered in the Western Ghats of India.MethodsA comprehensive sand fly survey was conducted across the Kollam, Thrissur, Idukki, Kasaragod and Malappuram districts of Kerala, India. The survey spanned both indoor and outdoor habitats using standard collection methods over a 3-year, 3-month period. DNA barcoding of samples was performed targeting mitochondrial cytochrome c oxidase subunit I (COI) gene, and the sequence generated was subjected to phylogenetic analysis.ResultsPhlebotomus (Anaphlebotomus) ajithii, a new sand fly species, is recorded and described in this communication. The morphological relationship of the new species to other members of the subgenus Anaphlebotomus is discussed. Mitochondrial COI barcode followed by phylogenetic analysis confirmed that specimens of Ph. ajithii belong to the same taxonomic group, while a genetic distance of 11.7% from congeners established it as a distinct species.ConclusionsThe Western Ghats, known for its rich biodiversity, has lacked systematic entomological surveys focusing on sand flies. This study aims to fill this gap and reports and describes a new species of sand fly.Graphical abstract

Cell cycle-driven transcriptome maturation confers multilineage competence to cardiopharyngeal progenitors.

During development, stem and progenitor cells divide and transition through germ layer- and lineage-specific multipotent states to generate the diverse cell types that compose an animal. Defined changes in biomolecular composition underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, multipotent cardiopharyngeal progenitors display multilineage transcriptional priming, whereby both the cardiac and pharyngeal muscle programs are partially active and coexist in the same progenitor cells, while their daughter cells engage in a cardiac or pharyngeal muscle differentiation path only after cell division. Here, using the tunicate Ciona, we studied the acquisition of multilineage competence and the coupling between fate decisions and cell cycle progression. We showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10(+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single cell RNA-seq (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S and G2 phases. We termed this process "transcriptome maturation", and identified candidate "mature genes", including the Rho GAP-coding gene Depdc1, which peak in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and proper oriented and asymmetric division that influences subsequent fate decisions, illustrating the concept of "behavioral competence". Both classic feedforward circuits and coupling with cell cycle progression drive transcriptome maturation, uncovering distinct levels of coupling between cell cycle progression and fateful molecular transitions. We propose that coupling competence and fate decision with the G2 and G1 phases, respectively, ensures the timely deployment of lineage-specific programs.

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Diet of moulting Swainson's Thrushes (Catharus ustulatus) and Tennessee Warblers (Leiothlypis peregrina) at a stopover site during fall migration measured with fecal DNA metabarcoding

Moult and migration are energetically demanding and require adequate nutrition. In some species, individuals may interrupt their fall migration to moult at discrete stopover locations outside of their breeding grounds (i.e., moult-migration) leading to competing nutritional demands for moult and migration. Here, we use DNA barcoding of fecal samples to compare the diet of moulting and actively migrating (post-moult) Swainson’s Thrushes (Catharus ustulatus) and Tennessee Warblers (Leiothlypis peregrina) during their fall migration stopover at a large urban greenspace in Montreal, Canada. Diet differed according to moult status, species, and seasonality. Swainson’s Thrushes had a broad diet with frequent detections of both insects and berry-producing shrubs; while detections in Tennessee Warblers’ diets were mainly arthropods. For both species, more actively migrating individuals consumed fleshy-fruiting plants than moulting individuals. A higher proportion of moulting birds consumed arthropods compared to active migrants, due to either arthropod availability or a dietary preference for proteinaceous foods to grow feathers. Both species and moult classes consumed more native plants than non-native plants later in the season. We show the importance of managing urban greenspaces with native plants and diverse food sources that can provide for the different dietary needs of migratory birds.

Morphological and molecular description of a new species of sandfly, Sergentomyia (Neophlebotomus) ashwanii sp. nov. (Diptera: Psychodidae) from Western Ghats, India

Objective: To report a new species of sandfly, Sergentomyia (Neophlebotomus) ashwanii sp. nov. (Diptera: Psychodidae) from Western Ghats, India. Methods: A systematic sandfly survey was conducted in the Thrissur and Kollam districts of Kerala, India using mechanical aspirators, light and sticky traps, both indoor and outdoor habitats, for a period of one year. Deoxyribonucleic acid barcoding of samples was performed targeting mitochondrial cytochrome oxidase I (COI) gene and sequence generated was subjected to phylogenetic analysis. Results: Sergentomyia (Neophlebotomus) ashwanii, a new sandfly species is recorded and described in this communication. A single row of 10-12 pointed teeth in the cibarium with 4-6 small denticles or fore-teeth are the key characteristics that is distinctive from other members of the subgenus Neophlebotomus. Mitochondrial COI barcode followed by phylogenetic analysis of the nucleotide sequence confirms that specimens of the species belong to the same taxonomic group while the genetic distance (14.2%) with the congeners established it to be a different species. Conclusions: The Western Ghats' being an important biodiversity hotspot and has dearth of systematic entomological surveys on sandflies. The current study tried to fill the void and also report a new sandfly species.

EDNA metabarcoding reveals a rich but threatened and declining elasmobranch community in West Africa’s largest marine protected area, the Banc d’Arguin

AbstractElasmobranchs (sharks and rays) are the most threatened marine vertebrates, particularly in tropical and subtropical areas. Their population status is often poorly understood due to insufficient information. Despite reportedly harbouring critical elasmobranch habitats, the Banc d’Arguin National Park (PNBA) in Mauritania lacks comprehensive and updated information on the diversity of elasmobranch species in the area. We developed a baseline inventory based on morphological and molecular identification and metabarcoding. DNA barcoding of tissue samples from elasmobranch processing sites and freshly sampled specimens was used to build a genetic reference database of local elasmobranch species. The richness and diversity of species in the PNBA were described via metabarcoding of seawater eDNA samples using an elasmobranch-specific assay and our reference database. We detected 27 species, including 12 new species records for the PNBA. We further uncover potentially undescribed species of Gymnura and Torpedo, while taxonomic corrections are noted for previously reported species. In particular, the reportedly abundant Mustelus mustelus was absent from tissue and eDNA samples, while M. punctulatus was detected instead. Taxa that have anecdotally become regionally extinct or rare (e.g., sawfishes, wedgefishes, lemon sharks) were not detected, highlighting local species diversity shifts within the last few decades. Results show that 67.9% of elasmobranch species in the PNBA are threatened with extinction according to the IUCN Red List of Threatened Species. This study emphasises the importance of taxonomic identification in support of species management and provides a baseline to inform future studies and conservation measures to avoid further species losses.

A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARS-CoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.

Validation of an Automated, Scalable Comprehensive Genomic Profiling Assay for Hematologic Malignancies

Introduction: The optimal diagnosis, prognosis, and treatment selection for hematologic disorders requires the assessment of somatic mutations across a subset of clinically relevant genes. Here we present the clinical validation results of a targeted next generation sequencing panel comprising all exons of 141 clinically relevant genes for myeloid and lymphoid malignancies. Methods: The panel was designed to include genes (n=141) based of clinical guidelines for myeloid diseases including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and lymphoid diseases including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and some lymphoma subtypes. The panel was validated to detect single nucleotide variants (SNVs), insertions/deletions (indels), FLT3 internal tandem duplications ( FLT3-ITDs), and sex. Gene level copy number alterations (CNAs) were validated for 11 genes and 5 sub-gene level CNAs in IKZF1, KMT2A, RUNX1, TET2, and TP53, and were selected based on clinical utility and frequency observed in a population of >10,000 microarray samples with confirmed myeloid or lymphoid indications. Custom hybridization probes (Twist Biosciences) were designed to interrogate all coding exons of the 141 genes using genomic libraries created from 250 ng of gDNA extracted from peripheral blood, bone marrow, or flow cytometry cell suspensions, followed by hybrid capture, and sequencing on Illumina DNA sequencers. Data analysis was performed using an in-house analysis pipeline to detect variants and perform rational filtering of technical artifacts. Validation studies were performed in a CAP/CLIA accredited clinical laboratory. Precision and concordance studies were performed using previously characterized clinical samples. SNVs, indels and FLT3-ITDs were orthogonally confirmed by an NGS-based laboratory developed test (ArcherDx) or by Sanger sequencing. CNAs were confirmed by digital multiplexed ligation-dependent probe amplification (dMLPA; MRC Holland). Results: Assay precision was determined using 15 previously characterized clinical samples with 36 SNVs with a variant allele frequency (VAF) range of 5.7-91.4%, 11 indels with a VAF range of 6.2-79.7%, one 24bp FLT3-ITD with a VAF of 30.2%, 17 deletions with a copy number (CN) range of 0.53-0.83, and 7 gains with CN range of 1.32-1.66. Three replicates of each sample were assessed for precision with 100% concordance observed for SNVs (36/36), indels (11/11), FLT3-ITD (1/1), CNAs (24/24), and gender (15/15). Inter-assay precision was assessed using multiple operators, instruments, reagent lots, and sample barcodes with the clinical samples. Interim analysis of concordance performed on 130 clinical samples demonstrated a positive percent agreement (PPA) of 99.7% for SNVs (334/335 in 114 clinical samples), 97.7% for indels (146/151 in 85 clinical samples), 98.7% for CNAs (30/30 in 17 clinical samples), and 96.7% for sex (127/130 in 130 clinical samples). Dilution series of both cell lines and clinical samples that will be used to determine analytical sensitivity are underway. Conclusions: This study highlights the clinical validation of a 141 gene NGS assay for the detection of clinically informative genomic alterations in hematologic malignancies. The detection of CNAs by NGS may be used to additionally confirm or monitor abnormalities previously detected by cytogenetics/microarray or inform the need for additional testing. Results from this validation study, when complete, will include at least 480 samples plus orthogonal testing. These data describe the performance of an assay to enable a comprehensive evaluation of genomic alterations using a single sample, further facilitating the use of broad NGS assays in hematologic malignancies.

HycDemux: a hybrid unsupervised approach for accurate barcoded sample demultiplexing in nanopore sequencing

DNA barcodes enable Oxford Nanopore sequencing to sequence multiple barcoded DNA samples on a single flow cell. DNA sequences with the same barcode need to be grouped together through demultiplexing. As the number of samples increases, accurate demultiplexing becomes difficult. We introduce HycDemux, which incorporates a GPU-parallelized hybrid clustering algorithm that uses nanopore signals and DNA sequences for accurate data clustering, alongside a voting-based module to finalize the demultiplexing results. Comprehensive experiments demonstrate that our approach outperforms unsupervised tools in short sequence fragment clustering and performs more robustly than current state-of-the-art demultiplexing tools for complex multi-sample sequencing data.

Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses

Assays linking cellular phenotypes with Tcell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between Tcell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After exvivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4+ and CD8+ populations. Combining complementary patient-matched single-cell RNA sequencing (scRNA-seq) data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility.

Hybrid Macromolecular Constructs as a Platform for Spectral Nanoprobes for Advanced Cellular Barcoding in Flow Cytometry.

Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.

Rapid Single-Round Pool Testing of Infectious Disease Enabled by Multicolor Digital Melting PCR.

Pooled nucleic acid amplification test is a promising strategy to reduce cost and resources for screening large populations for infectious disease. However, the benefit of pooled testing is reversed when disease prevalence is high, because of the need to retest each sample to identify infected individual when a pool is positive. Split, Amplify, and Melt analysis of Pooled Assay (SAMPA) is presented, a multicolor digital melting PCR assay in nanoliter chambers that simultaneously identify infected individuals and quantify their viral loads in a single round of pooled testing. This is achieved by early sample tagging with unique barcodes and pooling, followed by single molecule barcode identification in a digital PCR platform using a highly multiplexed melt curve analysis strategy. The feasibility is demonstrated of SAMPA for quantitative unmixing and variant identification from pools of eight synthetic DNA and RNA samples corresponding to the N1 gene, as well as from heat-inactivated SARS-CoV-2 virus. Single round pooled testing of barcoded samples with SAMPA can be a valuable tool for rapid and scalable population testing of infectious disease.

Abstract 5142: 50-parameter flow cytometry by CyTOF empowers comprehensive single-cell immune profiling of pulmonary immunosenescence in aged mice

Abstract High-parameter flow cytometry is essential for human and mouse studies to discover novel immunological mechanisms of cancer, infections, and immunosenescence. It plays an increasingly important role in cancer research to ensure clinical therapeutic success but is limited by the large amount of cell samples needed for staining controls. CyTOF® technology has transformed flow cytometry by enabling 50-plus-marker analysis per tube of sample, with easy panel design and no need for single-stained or autofluorescence controls. Flow cytometry by CyTOF provides an efficient and unbiased approach to discovering novel subsets and unique functional states of immune cells, maximizing insights from precious samples. Immunosenescence perturbs lung cancer onset and development, yet the mechanisms remain largely unknown. To study the pulmonary immune populations in aged (75 weeks old) and young adult (6-8 weeks old) mice, we built a 50-parameter panel (2 for single live-cell identification, 6 for live-cell sample barcoding, and 42 for immune profiling). A core panel of 32 antibodies was selected from Standard BioTools™ catalogs to detect key cell lineage and functional surface markers. A complementary panel with 10 Maxpar® antibodies was added to further study functional cell states. Maxpar Pathsetter™ software was used to create an automated analytical model for high-dimensional analysis. The 50-parameter panel successfully identified over 30 lymphoid and myeloid cell subsets including but not limited to T cells, B cells, NK cells, alveolar macrophages (AMs), dendritic cells, and neutrophils. The panel enabled high-fidelity detection of over 15 functional markers mediating proliferation, activation, inhibition, migration, tissue residence, and cellular metabolism. Automated in-depth analysis by Maxpar Pathsetter efficiently identified many aging-associated alterations in cell frequencies and functional states such as the enrichment of PD-1+ T cells and CD27- γδ T cells that could potentially perturb anti-tumor immunity. Moreover, CyTOF technology is uniquely advanced in characterizing autofluorescent cells such as AMs. A pro-inflammatory state (higher expression of MHC-II, CD80, and PD-L1) was specifically defined for AMs in aged lungs. This study demonstrates comprehensive single-cell immune profiling of mouse tissues with the products and solutions provided by Standard BioTools. Both the 50-parameter panel and Maxpar Pathsetter analytical model can be customized for deep characterization of specific immune populations according to the requirements of various cancer studies in mice. By utilizing end-to-end solutions offered by Standard BioTools, flow cytometry by CyTOF can significantly facilitate the mechanistic studies of mouse models to expand the understanding of human cancers and accelerate therapeutic development. Citation Format: Wenxi Xu, Stephen Li, Alexandre Bouzekri, Lauren Tracey, Christina Loh. 50-parameter flow cytometry by CyTOF empowers comprehensive single-cell immune profiling of pulmonary immunosenescence in aged mice. [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 5142.

BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples.

The ability to profile transcriptomes and characterize global gene expression changes has been greatly enabled by the development of RNA sequencing technologies (RNA-seq). However, the process of generating sequencing-compatible cDNA libraries from RNA samples can be time-consuming and expensive, especially for bacterial mRNAswhich lack poly(A)-tails that are often used to streamline this process for eukaryotic samples. Compared to the increasing throughput and decreasing cost of sequencing, library preparation has had limited advances. Here, we describe bacterial-multiplexed-seq (BaM-seq), an approach that enables simple barcoding of many bacterial RNA samples that decreases the time and cost of library preparation. We also present targeted-bacterial-multiplexed-seq (TBaM-seq) that allows for differential expression analysis of specific gene panels with over 100-fold enrichment in read coverage. In addition, we introduce the concept of transcriptome redistribution based on TBaM-seq that dramatically reduces the required sequencing depth while still allowing for quantification of both highly and lowly abundant transcripts. These methods accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches. Together, use of these library preparation protocols allows for fast, affordable generation of sequencing libraries.

The Cultural and Commercial Value of Tulsi (Ocimum tenuiflorum L.): Multidisciplinary Approaches Focusing on Species Authentication.

Tulsi (Holy basil, Ocimum tenuiflorum L., Lamiaceae), native to Asia, has become globalised as the cultural, cosmetic, and medicinal uses of the herb have been popularised. DNA barcoding, a molecular technique used to identify species based on short regions of DNA, can discriminate between different species and identify contaminants and adulterants. This study aimed to explore the values associated with Tulsi in the United Kingdom (UK) and authenticate samples using DNA barcoding. A mixed methods approach was used, incorporating social research (i.e., structured interviews) and DNA barcoding of Ocimum samples using the ITS and trnH-psbA barcode regions. Interviews revealed the cultural significance of Tulsi: including origins, knowledge exchange, religious connotations, and medicinal uses. With migration, sharing of plants and seeds has been seen as Tulsi plants are widely grown in South Asian (SA) households across the UK. Vouchered Ocimum specimens (n = 33) were obtained to create reference DNA barcodes which were not available in databases. A potential species substitution of O. gratissimum instead of O. tenuiflorum amongst SA participants was uncovered. Commercial samples (n = 47) were difficult to authenticate, potentially due to DNA degradation during manufacturing processes. This study highlights the cultural significance of Tulsi, despite a potential species substitution, the plant holds a prestigious place amongst SA families in the UK. DNA barcoding was a reliable way to authenticate Ocimum species.

Phylogenetic Relationships of Grammitid Fern Diversity of Gunung Tama Abu (Sarawak)

The forests of Borneo are famous for their plant diversity, but this diversity is seriously threatened. Despite remarkable progress, the taxonomic status and geographic distribution of many species have not been clarified. Here, we explore the use of DNA barcoding to improve not only the recording of local species diversity, but also our understanding of the fern Tree-of-Life. We focused specifically on grammitid ferns collected at Gunung Tama Abu in Sarawak. The four new samples comprised three species of which two were studied using molecular phylogenetics for the first time. These three species were found to be nested in clades corresponding to the established generic taxonomy. The two sampled accessions of Acrosorus streptophyllus formed a sister clade to A. friderici-et-pauli. By increasing the inclusion of Acrosorus species from one to two in the assembled phylogenetic hypothesis, our results provide support to the monophyly of this genus based on DNA sequence data for the first time. The newly sampled Borneo accession of Scleroglossum pyxidatum was sister to a clade comprising accessions of S. sulcatum, whereas the sample of Prosaptia alata formed a clade together with other accessions of this species. In conclusion, DNA barcoding of samples obtained during fieldwork in remote but species rich places enhance the recording of the Tree-of-Life and enable the establishment of reliable DNA barcodes for species rich lineages.

Abstract 3908: A streamlined and automated approach to high-content cytometric immunophenotyping with CyTOF XT

Abstract High-parameter immune profiling is crucial in translational and clinical research to quantify changes in immune cell populations over time. CyTOF® mass cytometry is a high-plex, single-cell analysis platform that uses isotopically pure metal-labeled antibodies. The major advantage of CyTOF is its ability to resolve 40-plus markers in a single panel without compensation, making mass cytometry ideal for routine immunophenotyping. The autosampler module of CyTOF XT™ provides significant time savings by allowing automated sample acquisition. Tubes of pelleted stained samples are loaded into the Autosampler carousel, and the samples are resuspended with EQ™ Calibration Beads for acquisition. User input is only required during instrument startup, tuning, and batch setup. The added automation of CyTOF XT provides a streamlined workflow for suspension mass cytometry. Testing was performed to ensure that the data obtained on CyTOF XT was comparable to manual acquisition systems. The performance of CyTOF XT was tested in parallel with its predecessor, Helios™. Several workflows and applications for suspension mass cytometry including sample barcoding with the Cell-ID™ 20-Plex Pd Barcoding Kit, and surface, cytoplasmic, and nuclear staining and phosphostaining were evaluated on human PBMC. Manual gating analysis was performed to assess population frequencies and median intensities for each marker. Resolution index was calculated to assess how well positive and negative populations separated from each other. There was no significant difference between population frequencies analyzed between the two CyTOF systems. Moreover, samples acquired on CyTOF XT, on average, resulted in greater signal resolution between positive and negative populations compared to Helios. The Maxpar Direct Immune Profiling System was also compared on CyTOF XT and Helios using human whole blood and PBMC. The Maxpar® Direct™ Immune Profiling Assay™ and Maxpar Pathsetter™ software were developed as a sample-to-answer system for human immune profiling using CyTOF. The Maxpar Direct Immune Profiling Assay includes an optimized panel of 30 unique markers in a dry, single-tube format. Maxpar Pathsetter is an automated software used to report population statistics, stain assessments, and relevant data plots for the panel. The automated staining assessment in Maxpar Pathsetter was compared between files acquired on CyTOF XT and Helios. Comparable population frequencies were obtained between the two acquisition systems, and improved staining assessment was observed on CyTOF XT. Overall, these studies demonstrate that CyTOF XT generates better signal resolution as compared to Helios. The automated acquisition of CyTOF XT enables researchers to streamline immunophenotyping of human samples while accurately and reproducibly monitoring changes in immune cell subsets. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Thiru Selvanantham, Stephen K.H. Li, Nick Zabinyakov, Alexandre Bouzekri, Raymond Jong, Michael Sullivan, Alexander Laboda, Daniel Majonis, Christina Loh. A streamlined and automated approach to high-content cytometric immunophenotyping with CyTOF XT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3908.

Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction.

We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.