Systematic benchmark of demultiplexing workflows for direct RNA sequencing
Direct RNA nanopore sequencing offers the capacity to directly profile native RNAs, enabling detection of diverse epitranscriptomic modifications based on current changes as the RNA passes through a pore. Innovations in chemistry, hardware and bioinformatic tools have increased sequencing capacity and accuracy, creating demand for sequencing multiple samples within individual runs. Two recent…
A systematic evaluation of demultiplexing workflows for direct RNA sequencing has been conducted, comparing two solutions: WarpDemuX and SeqTagger. Both methods demonstrate high precision in demultiplexing, but their performance varies in key aspects. SeqTagger consistently performs well when used with its recommended settings, while WarpDemuX's performance fluctuates depending on the model used, barcode design, and read quality.
An interesting finding from this study is the use of IVT-derived score cutoffs, which allows for calibration of the dataset, increasing WarpDeMux assignment yield. However, precision achieved on these internal controls (IVTs) may not always be consistent in species reads. The results emphasize the importance of matched IVTs as internal controls in determining demultiplexing settings and highlight the necessity to validate demultiplexing precision in biological RNA populations.
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