An ancestral SARS-CoV-2 vaccine induces anti-Omicron variants antibodies by hypermutation.

The immune escape of Omicron variants significantly subsides by the third dose of an mRNA vaccine. However, it is unclear how Omicron variant-neutralizing antibodies develop under repeated vaccination. We analyze blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyze their B-cell receptor (BCR) repertoires at six time points in total. The concomitant reactivity to both ancestral and Omicron receptor-binding domain (RBD) is achieved by a limited number of BCR clonotypes depending on the accumulation of somatic hypermutation (SHM) after the third dose. Our findings suggest that SHM accumulation in the BCR space to broaden its specificity for unseen antigens is a counterprotective mechanism against virus variant immune escape.

Stereotypic T cell receptor clonotypes in the thymus and peripheral blood of Myasthenia gravis patients.

Myasthenia Gravis (MG) patients with anti-acetylcholine receptor (AChR) antibodies frequently show hyperplastic thymi with ectopic germinal centers, where autoreactive B cells proliferate with the aid of T cells. In this study, thymus and peripheral blood (PB) samples were collected from ten AChR antibody-positive MG patients. T cell receptor (TCR) repertoires were analyzed using next-generation sequencing (NGS), and compared with that of an age and sex matched control group generated from a public database. Certain V genes and VJ gene recombination pairs were significantly upregulated in the TCR chains of αß-T cells in the PB of MG patients compared to the control group. Furthermore, the TCR chains found in the thymi of MG patients had a weighted distribution to longer CDR3 lengths when compared to the PB of MG patients, and the TCR beta chains (TRB) in the MG group's PB showed increased clonality encoded by one upregulated V gene. When TRB sequences were sub-divided into groups based on their CDR3 lengths, certain groups showed decreased clonality in the MG group's PB compared to the control group's PB. Finally, we demonstrated that stereotypic MG patient-specific TCR clonotypes co-exist in both the PB and thymi at a much higher frequency than that of the clonotypes confined to the PB. These results strongly suggest the existence of a biased T cell-mediated immune response in MG patients, as observed in other autoimmune diseases.

Highly Accurate Sequence- and Position-Independent Error Profiling of DNA Synthesis and Sequencing.

A comprehensive error analysis of DNA-stored data during processing, such as DNA synthesis and sequencing, is crucial for reliable DNA data storage. Both synthesis and sequencing errors depend on the sequence and the transition of bases of nucleotides; ignoring either one of the error sources leads to technical challenges in minimizing the error rate. Here, we present a methodology and toolkit that utilizes an oligonucleotide library generated from a 10-base-shifted sequence array, which is individually labeled with unique molecular identifiers, to delineate and profile DNA synthesis and sequencing errors simultaneously. This methodology enables position- and sequence-independent error profiling of both DNA synthesis and sequencing. Using this toolkit, we report base transitional errors in both synthesis and sequencing in general DNA data storage as well as degenerate-base-augmented DNA data storage. The methodology and data presented will contribute to the development of DNA sequence designs with minimal error.

Canine Somatic Mutations from Whole-Exome Sequencing of B-Cell Lymphomas in Six Canine Breeds-A Preliminary Study.

Canine lymphoma (CL) is one of the most common malignant tumors in dogs. The cause of CL remains unclear. Genetic mutations that have been suggested as possible causes of CL are not fully understood. Whole-exome sequencing (WES) is a time- and cost-effective method for detecting genetic variants targeting only the protein-coding regions (exons) that are part of the entire genome region. A total of eight patients with B-cell lymphomas were recruited, and WES analysis was performed on whole blood and lymph node aspirate samples from each patient. A total of 17 somatic variants (GOLIM4, ITM2B, STN1, UNC79, PLEKHG4, BRF1, ENSCAFG00845007156, SEMA6B, DSC1, TNFAIP1, MYLK3, WAPL, ADORA2B, LOXHD1, GP6, AZIN1, and NCSTN) with moderate to high impact were identified by WES analysis. Through a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 17 genes with somatic mutations, a total of 16 pathways were identified. Overall, the somatic mutations identified in this study suggest novel candidate mutations for CL, and further studies are needed to confirm the role of these mutations.

Barcoded multiple displacement amplification for high coverage sequencing in spatial genomics.

Determining mutational landscapes in a spatial context is essential for understanding genetically heterogeneous cell microniches. Current approaches, such as Multiple Displacement Amplification (MDA), offer high genome coverage but limited multiplexing, which hinders large-scale spatial genomic studies. Here, we introduce barcoded MDA (bMDA), a technique that achieves high-coverage genomic analysis of low-input DNA while enhancing the multiplexing capabilities. By incorporating cell barcodes during MDA, bMDA streamlines library preparation in one pot, thereby overcoming a key bottleneck in spatial genomics. We apply bMDA to the integrative spatial analysis of triple-negative breast cancer tissues by examining copy number alterations, single nucleotide variations, structural variations, and kataegis signatures for each spatial microniche. This enables the assessment of subclonal evolutionary relationships within a spatial context. Therefore, bMDA has emerged as a scalable technology with the potential to advance the field of spatial genomics significantly.

I-LIFT (image-based laser-induced forward transfer) platform for manipulating encoded microparticles.

Encoded microparticles have great potential in small-volume multiplexed assays. It is important to link the micro-level assays to the macro-level by indexing and manipulating the microparticles to enhance their versatility. There are technologies to actively manipulate the encoded microparticles, but none is capable of directly manipulating the encoded microparticles with homogeneous physical properties. Here, we report the image-based laser-induced forward transfer system for active manipulation of the graphically encoded microparticles. By demonstrating the direct retrieval of the microparticles of interest, we show that this system has the potential to expand the usage of encoded microparticles.

Spatial epitranscriptomics reveals A-to-I editome specific to cancer stem cell microniches.

Epitranscriptomic features, such as single-base RNA editing, are sources of transcript diversity in cancer, but little is understood in terms of their spatial context in the tumour microenvironment. Here, we introduce spatial-histopathological examination-linked epitranscriptomics converged to transcriptomics with sequencing (Select-seq), which isolates regions of interest from immunofluorescence-stained tissue and obtains transcriptomic and epitranscriptomic data. With Select-seq, we analyse the cancer stem cell-like microniches in relation to the tumour microenvironment of triple-negative breast cancer patients. We identify alternative splice variants, perform complementarity-determining region analysis of infiltrating T cells and B cells, and assess adenosine-to-inosine base editing in tumour tissue sections. Especially, in triple-negative breast cancer microniches, adenosine-to-inosine editome specific to different microniche groups is identified.

Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides.

Synthesizing engineered bacteriophages (phages) for human use has potential in various applications ranging from drug screening using a phage display to clinical use using phage therapy. However, the engineering of phages conventionally involves the use of an in vivo system that has low production efficiency because of high virulence against the host and low transformation efficiency. To circumvent these issues, de novo phage genome synthesis using chemically synthesized oligonucleotides (oligos) has increased the potential for engineering phages in a cell-free system. Here, we present a cell-free, low-cost, de novo gene synthesis technology called Sniper assembly for phage genome construction. With massively parallel sequencing of microarray-synthesized oligos, we generated and identified approximately 100 000 clonal DNA clusters in vitro and 5000 error-free ones in a cell-free environment. To demonstrate its practical application, we synthesized the Acinetobacter phage AP205 genome (4268 bp) using 65 sequence-verified DNA clones. Compared to previous reports, Sniper assembly lowered the genome synthesis cost ($0.0137/bp) by producing low-cost sequence-verified DNA.