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bioRxiv · 10.1101/2025.05.30.657070

Synthbar: A Lightweight Tool for Adding Synthetic Barcodes to Sequencing Reads

Morrison, J.·Johnson, B. K.·Shen, H.
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Abstract

Preparation of single-cell sequencing libraries includes adding nucleotide barcodes to assist with pooling samples or cells together for sequencing. The popularity of droplet-based single-cell protocols has spurred the development of computational tools that expect the read structure of the assay to include a cell barcode (CB). Microwell plate-based protocols, such as the Switching Mechanism At the 5 end of the RNA Transcript (SMART) single-cell RNA sequencing (scRNA-seq) family of methods, typically do not add a CB as part of the library preparation method as there is typically one cell per well and standard unique dual indices are sufficient for multiplexing. While several tools exist to manipulate and parse varying single-cell read structures, no tool is currently available to easily add synthetic CBs to enable use of computational tooling that expects the presence of a CB, such as STARsolo, zUMIs, and Alevin. Synthbar fills this gap as a lightweight tool that is assay agnostic, can add user-defined CBs, and modify read structures. Availability and ImplementationSource code and binaries are freely available at https://github.com/jamorrison/synthbar under the MIT license. Synthbar is implemented in C and is supported on macOS and Linux. Supplementary informationSupplementary data are available at the end of the document.

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Morrison, J., Johnson, B. K., Shen, H.. 2025-06-02. Synthbar: A Lightweight Tool for Adding Synthetic Barcodes to Sequencing Reads. https://doi.org/10.1101/2025.05.30.657070

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