Genetic demultiplexing and transcript start site identification from nanopore sequencing of 10x Genomics multiome libraries

Abstract Short-read Illumina sequencing of 10x Genomics single-nucleus multiome libraries captures only the 3’ end of RNA transcripts, losing transcription start site (TSS) information. Here we demonstrate nanopore sequencing of 10x multiome libraries, which enables the profiling of full length transcripts. We show concordance with common short-read sequencing based workflows including successful genetic demultiplexing of nanopore data despite its higher error rate. We compare TSS identified using nanopore sequencing of multiome cDNA to those identified using a short-read 5’ assay, and provide an optimized approach for the preprocessing of nanopore reads prior to TSS identification. We find that nanopore sequencing of multiome cDNA captures a median of 63% of the TSS detected by the 5’ assay. Competing Interest Statement All authors received funding from Pfizer, Inc. for this work. M. Piper, E. Pashos, P. Jean, E.B. Fauman, F. Damilano, and R.J. Roth Flach are or were employees and/or stockholders of Pfizer Footnotes Reordered document to move figure legends