Abstract: Provided herein are methods, compositions, and kits for simultaneously sequencing polynucleotides from a plurality of samples in a single sequencing run. In an embodiment, the present invention improves efficiency of the next-generation sequencing process, in part, by reducing reaction volumes to a sub-microliter range and generating and using a set of novel barcode sequences to tag a plurality of polynucleotides. In addition, the sample preparation processes have been simplified to save time and cost, while providing high-quality sequence coverage for all samples.