First records of three genera, Cyphomella Sæther, 1977, Olecryptotendipes Zorina, 2007 and Robackia Sæther, 1977 of the Harnischia complex from India with description of O. extentus sp. n., O. obtunsus sp. n. and R. aequilongia sp. n. (Diptera: Chironomidae)

Three genera, Cyphomella Sæther, 1977, Olecryptotendipes Zorina, 2007 and Robackia Sæther, 1977, of the Harnischia complex are recorded for the first time from India. Two new species of Olecryptotendipes and one new species of Robackia are described on the basis of adult males. Cyphomella camelus (Kieffer, 1955), described from Afrotropical and Palaearctic regions, is recorded for the first time from India and its description is supplemented. The molecular barcodes of three species, O. obtunsus sp. n., C. camelus and R. aequilongia sp. n., are also provided. Revised world keys to the adult males of the Olecryptotendipes and Robackia are also provided.

Phylogenomic and morphological relationships among the botryllid ascidians (Subphylum Tunicata, Class Ascidiacea, Family Styelidae)

Ascidians (Phylum Chordata, Class Ascidiacea) are a large group of invertebrates which occupy a central role in the ecology of marine benthic communities. Many ascidian species have become successfully introduced around the world via anthropogenic vectors. The botryllid ascidians (Order Stolidobranchia, Family Styelidae) are a group of 53 colonial species, several of which are widespread throughout temperate or tropical and subtropical waters. However, the systematics and biology of this group of ascidians is not well-understood. To provide a systematic framework for this group, we have constructed a well-resolved phylogenomic tree using 200 novel loci and 55 specimens. A Principal Components Analysis of all species described in the literature using 31 taxonomic characteristics revealed that some species occupy a unique morphological space and can be easily identified using characteristics of adult colonies. For other species, additional information such as larval or life history characteristics may be required for taxonomic discrimination. Molecular barcodes are critical for guiding the delineation of morphologically similar species in this group.

Development of a program for in silico optimized selection of oligonucleotide-based molecular barcodes

Short DNA oligonucleotides (~4 mer) have been used to index samples from different sources, such as in multiplex sequencing. Presently, longer oligonucleotides (8–12 mer) are being used as molecular barcodes with which to distinguish among raw DNA molecules in many high-tech sequence analyses, including low-frequent mutation detection, quantitative transcriptome analysis, and single-cell sequencing. Despite some advantages of using molecular barcodes with random sequences, such an approach, however, makes it impossible to know the exact sequences used in an experiment and can lead to inaccurate interpretation due to misclustering of barcodes arising from the occurrence of unexpected mutations in the barcodes. The present study introduces a tool developed for selecting an optimal barcode subset during molecular barcoding. The program considers five barcode factors: GC content, homopolymers, simple sequence repeats with repeated units of dinucleotides, Hamming distance, and complementarity between barcodes. To evaluate a selected barcode set, penalty scores for the factors are defined based on their distributions observed in random barcodes. The algorithm employed in the program comprises two steps: i) random generation of an initial set and ii) optimal barcode selection via iterative replacement. Users can execute the program by inputting barcode length and the number of barcodes to be generated. Furthermore, the program accepts a user’s own values for other parameters, including penalty scores, for advanced use, allowing it to be applied in various conditions. In many test runs to obtain 100000 barcodes with lengths of 12 nucleotides, the program showed fast performance, efficient enough to generate optimal barcode sequences with merely the use of a desktop PC. We also showed that VFOS has comparable performance, flexibility in program running, consideration of simple sequence repeats, and fast computation time in comparison with other two tools (DNABarcodes and FreeBarcodes). Owing to the versatility and fast performance of the program, we expect that many researchers will opt to apply it for selecting optimal barcode sets during their experiments, including next-generation sequencing.

UMI-linked nanopore consensus sequencing (UMIC-seq) of highly similar gene variants

Here we present a straightforward unique molecular identifier (UMI)-linked nanopore consensus sequencing workflow (UMIC-seq), resulting in cost-effective and accurate long-read sequencing of amplicons. Short random molecular barcodes (i.e. unique molecular identifiers, UMIs) are attached to a pool of gene variants prior to nanopore sequencing to enable reliable clustering and the generation of accurate consensus sequences, even when starting from highly similar gene variants (e.g. a library of point mutants in directed protein evolution) that could not be reliably distinguished in the ordinary nanopore sequencing output.

Novel Method for Clonal Selection of Multiplexed Engineered CAR-T Cells Which Uniquely Demonstrate Anti-Tumor Functionality in the Tumor Microenvironment

Despite the success of chimeric antigen receptor (CAR)-T cell therapy in various hematologic malignancies, obstacles to an effective therapeutic outcome are highly dependent on the tumor type being targeted and the immune microenvironment that the CAR-T cells encounter. For example, the presence of suppressive cells and soluble factors in the tumor microenvironment (TME) can prevent continued antitumor function of CAR-T cells. Toward this end, we explored multiple genetic editing options, including IL15-based edits, for improving the persistence and activation state of CAR-T cells in the TME. CAR-T cells engineered to express one of five different molecular barcoded constructs were developed and compared, including two versions of an IL-15 signaling complex (IL15RF), constitutively active IL-7 receptor (ca-IL7R), IL-21 signaling complex (IL21RF), and CD16 transgenes. The use of molecular tags allowed us to track CAR-T cell subpopulations in a complex pool with great resolution via next-generation sequencing (NGS) technology. Subsequent in vitro functional testing was performed to assess CAR-T expansion and function in response to serial stimulation with tumor cells bearing cognate antigen. Results showed that after four rounds of stimulation, cytotoxicity was enhanced in CAR-T cells engineered with the ca-IL7R and IL15RF transgene edits (1.5-fold increase in target cell lysis compared to control). Furthermore, an increased proportion of IL-2 producing cells was seen in CAR-T cells expressing the ca-IL7R and IL15RF-based edits (2-fold increase compared to control). In the initial proof of concept study, the best expansion after eight rounds of stimulation was seen in CAR-Ts engineered with IL15RF-based edits. Furthermore, using NGS to screen for the unique molecular barcodes in the CAR-T cell pool, we confirmed the enrichment of CAR-T cells with IL15RF-based edits over multiple rounds of stimulation. Single cell RNAseq was also performed after four and eight rounds of stimulation, where multiple clusters of CAR-T cells were identified and traced back to performance in vitro. Analysis of single cell clusters without IL15RF-based edits exhibited an increase in expression of the checkpoint receptor CTLA4 (p = 4.2E-2) and transcription factor GATA3 (p = 6.9E-5), while clusters with IL15RF-based edits had increased expression of effector molecules GZMB (p = 3.6E-2) and GZMH (p = 2.9E-8), T cell memory related markers CD62L (p = 5.2E-3) and CD27 (p = 2.2E-6), as well as increased expression of the cell proliferation marker Ki-67 (p = 3.3E-12). Because the presence and expansion of T cells in the tumor can be a good prognostic indicator for response to therapy, we used the pool of barcoded CAR-T cells and tested for enrichment/infiltration in a subcutaneous solid tumor implanted in NSG mice. Importantly, enrichment for CAR-T cells with IL15RF-based edits was observed using an NGS readout for the molecular barcodes present in the tumors. Analysis of the data from spatial transcriptomics on tumor sections, and single cell RNAseq of dissociated tumor samples, further informed our understanding of how CAR-T cells with IL15-based edits performed better in the TME (4-fold increase compared to control). The strategy of using molecular barcoded constructs for evaluating clonal populations of engineered CAR-T cells in a pool is shown here to be feasible and that it can be applied as a precise method to concurrently screen many distinct engineered modalities to improve effector cell function, homing and residence in various solid tumor settings. Disclosures Peralta: Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. Lu:Fate Therapeutics, Inc.: Current Employment. Yao:Fate Therapeutics, Inc.: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Sui:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Shoemaker:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Short Communication: Molecular barcode and morphology analysis of Malva pseudolavatera Webb & Berthel and Malva sylvestris L. from Ecuador

Sarmiento-Tomalá G, Santos-Ordóñez E, Miranda-Martínez M, Pacheco-Coello R, Scull-Lizama R, Gutiérrez-Gaitén Y, Delgado-Hernández R. 2020. Short Communication: Molecular barcode and morphology analysis of Malva pseudolavatera Webb & Berthel and Malva sylvestris L from Ecuador. Biodiversitas 21: 3554-3560. In Ecuador, several plant species are used in traditional medicine without a criterion of family, genera, or chemical composition. The species of the genus Malva (Malva pseudolavatera Webb & Berthel and Malva sylvestris L), introduced in Ecuador, are widely used by the population; however, unlike the species M. sylvestris, for M. pseudolavatera there is no information about its composition and properties. Plant material was collected in the province of Chimborazo in Ecuador and taxonomic classification was performed. Histological study was performed in leaves and powder drug. Molecular barcodes were generated using the ribulose bisphosphate carboxylase large chain (rbcL), maturase K (matK), internal transcribed spacer 1 (ITS1) and ITS2 sequences. Micro-morphological analysis revealed that no major structural differences were observed between the two species. Sequence analysis of molecular barcodes revealed that samples of the different species showed a close relation to each other due to the high percentage of similarity. The ITS sequences showed that the two samples correspond to different species of Malva; while for the rbcL and matK, interspecies differentiation could not be detected. Therefore, ITS could be used for interspecific analysis.

Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador

Background Mimusops coriacea (A.DC.) Miq., (Sapotaceae), originated from Africa, were introduced to coastal areas in Ecuador where it is not extensively used as a traditional medicine to treat various human diseases. Different therapeutically uses of the species include: analgesic, antimicrobial, hypoglycemic, inflammation and pain relieve associated with bone and articulation-related diseases. Furthermore, Mimusops coriacea could be used as anti-oxidant agent. However, botanical, chemical or molecular barcode information related to this much used species is not available from Ecuador. In this study, morphological characterization was performed from leaves, stem and seeds. Furthermore, genetic characterization was performed using molecular barcodes for rbcL, matk, ITS1 and ITS2 using DNA extracted from leaves. Methods Macro-morphological description was performed on fresh leaves, stem and seeds. For anatomical evaluation, tissues were embedded in paraffin and transversal dissections were done following incubation with sodium hypochlorite and safranin for coloration and fixated later in glycerinated gelatin. DNA extraction was performed using a modified CTAB protocol from leaf tissues, while amplification by PCR was accomplished for the molecular barcodes rbcL, matK, ITS1 and ITS2. Sequence analysis was performed using blast in the GenBank. Phylogenetic analysis was performed with accessions queried in the GenBank belonging to the subfamily Sapotoideae. Results Leaf size was 13.56 ± 1.46 × 7.49 ± 0.65 cm; where is a macro-morphological description of the stem (see Methods). The peel of the seeds is dark brown. Sequence analysis revealed that amplicons were generated using the four barcodes selected. Phylogenetic analysis indicated that the barcodes rbcL and matK, were not discriminated between species within the same genus of the subfamily Sapotoideae. On the other hand, the ITS1 and ITS2 were discriminative at the level of genus and species of the Sapotoideae.

A new normalization for Nanostring nCounter gene expression data

The Nanostring nCounter gene expression assay uses molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. These counts need to be normalized to adjust for the amount of sample, variations in assay efficiency and other factors. Most users adopt the normalization approach described in the nSolver analysis software, which involves background correction based on the observed values of negative control probes, a within-sample normalization using the observed values of positive control probes and normalization across samples using reference (housekeeping) genes. Here we present a new normalization method, Removing Unwanted Variation-III (RUV-III), which makes vital use of technical replicates and suitable control genes. We also propose an approach using pseudo-replicates when technical replicates are not available. The effectiveness of RUV-III is illustrated on four different datasets. We also offer suggestions on the design and analysis of studies involving this technology.

Exploiting Molecular Barcodes in High-Throughput Cellular Assays

Multiplexing strategies, which greatly increase the number of simultaneously measured parameters in single experiments, are now being widely implemented by both the pharmaceutical industry and academic researchers. Color has long been used to identify biological signals and, when combined with molecular barcodes, has substantially enhanced the depth of multiplexed sample characterization. Moreover, the recent advent of DNA barcodes has led to an explosion of innovative cell sequencing approaches. Novel barcoding strategies also show great promise for encoding spatial information in transcriptomic studies, and for precise assessment of molecular abundance. Both color- and DNA-based barcodes can be conveniently analyzed with either a microscope or a cytometer, or via DNA sequencing. Here we review the basic principles of several technologies used to create barcodes and detail the type of samples that can be identified with such tags.

The first smut fungus, Thecaphora anthemidis sp. nov. (Glomosporiaceae), described from Anthemis (Asteraceae)

There are 63 known species ofThecaphora(Glomosporiaceae, Ustilaginomycotina), a third of which occur on Asteraceae. These smut fungi produce yellowish-brown to reddish-brown masses of spore balls in specific, mostly regenerative, plant organs. A species ofThecaphorawas collected in the flower heads ofAnthemischia(Anthemideae, Asteraceae) on Rhodes Island, Greece, in 2015 and 2017, which represents the first smut record of a smut fungus on a host plant species in this tribe. Based on its distinctive morphology, host species and genetic divergence, this species is described asThecaphoraanthemidissp. nov.Molecular barcodes of the ITS region are provided for this and several other species ofThecaphora. A phylogenetic and morphological comparison to closely related species showed thatTh.anthemidisdiffered from other species ofThecaphora.Thecaphoraanthemidisproduced loose spore balls in the flower heads and peduncles ofAnthemischiaunlike other flower-infecting species.