High-content single-cell combinatorial indexing

Single-cell combinatorial indexing (sci) with transposase-based library construction increases the throughput of single-cell genomics assays but produces sparse coverage in terms of usable reads per cell. We develop symmetrical strand sci (‘s3’), a uracil-based adapter switching approach that improves the rate of conversion of source DNA into viable sequencing library fragments following tagmentation. We apply this chemistry to assay chromatin accessibility (s3-ATAC) in human cortical and mouse whole brain tissues, with mouse datasets demonstrating a 6-to-13-fold improvement in usable reads per cell compared with other available methods. Application of s3 to single-cell whole genome sequencing (s3-WGS) and to whole genome plus chromatin conformation (s3-GCC) yields 148 and 14.8 fold improvements, respectively, in usable reads per cell compared with sci-DNA-seq and sci-HiC. We show that s3-WGS and s3-GCC resolve subclonal genomic alterations in patient-derived pancreatic cancer cell lines. We expect that the s3 platform will be compatible with other transposase-based techniques, including sci-MET or CUT&Tag.