Denis Syrko

Single-cell RNA sequencing (scRNA-Seq) provides valuable insights into cell biology. However, current scRNA-Seq analytic approaches do not distinguish between spliced and unspliced mRNA at the level of dimensionality reduction. RNA velocity paradigm suggests that the presence of unspliced mRNA reflects transitional cell states, informative for studies of dynamic processes such as embryogenesis or tissue regeneration. Alternatively, stable cell subsets may also maintain translationally repressed spliced mRNA (e.g., in P-bodies) and/or unspliced mRNA reservoirs for prompt initiation of transcription-independent expression. Thus, functional cell subsets may differ not only in the current levels of actively produced mRNAs, but also in which mRNAs and in what forms are stored in the nucleus and cytoplasm. To enable splicing-aware analysis of scRNA-Seq data, we developed a method called SANSARA (Splicing-Aware scrNa-Seq AppRoAch). We employed SANSARA to characterize peripheral blood regulatory T cell (T _reg ) subsets, revealing a complementary interplay between the FOXP3 and Helios master transcription factors and high levels of spliced IL10RA , LGALS3 , FCRL3 , CD38 , ITGAL , and LEF1 mRNAs in effector T _reg s. Among Th1 and cytotoxic CD4 ⁺ T cell subsets, SANSARA also revealed substantial splicing heterogeneity across subset-specific genes. SANSARA is straightforward to implement in current data analysis pipelines and opens new dimensions for scRNA-Seq-based discoveries.

The MGI (MGI Tech Co. Ltd., China) next-generation sequencing platform, including the DNBSEQ-G50, -G400, and -T7 sequencers, is being actively adopted in research. Despite its widespread adoption, challenges persist in the form of limitations associated with the manufacturer's provided barcode set for library preparation. These limitations include constraints on the number of samples that can be concurrently sequenced, compatibility issues with barcodes from diverse or incomplete sets, and restrictions on the sample ratio. Purpose: to develop a universal method that allows sequencing of up to 252 samples simultaneously on a single sequencer lane, while eliminating barcode-related limitations. We proposed a “quad method” that provides 4 or 4n+2 equilibration of barcodes. This paper also delves into its comprehensive analysis, verification procedures, seamless integration into the sequencing process and validation of the method on the DNBSEQ G-400 platform. The quad method showed efficiency and reliability, allowing sequencing of up to 252 samples simultaneously without compromising data quality. The proposed method optimizes library preparation and improves the flexibility of sequencing on the MGI platform.

The recently launched MGI platform (DNBSEQ-G50, -G400 and -T7 sequencers) has been gaining popularity for next-generation sequencing. However, the barcode set for library preparation provided by the manufacturer has certain limitations on the number of samples that can be sequenced at a time and the compatibility of barcodes from different or incomplete sets as well as a ratio between the samples. In this paper, we present a protocol for designing custom barcodes expanding the pre-existing barcode set and demonstrate its performance on the MGI Tech Co. Ltd. (China) machines. We developed a universal “quad method” which allows for simultaneous sequencing of any number of samples up to 252 per lane which is multiple of 4 or 4n+2. Here, we describe this method, its analysis, verification, and integration into the sequencing as well as its validation for sequencing using the DNBSEQ G-400 machine.