Abstract: This document provides methods and materials for determining the identity of the terminal nucleotide (e.g., the 3? terminal nucleotide) of a nucleic acid molecule. For example, methods and materials for using RNA-DNA chimeric primers during a primer extension reaction followed by treatment with one or more RNAse enzymes to prepare nucleic acid molecules that can be analyzed to identify the terminal nucleotides added to the RNA-DNA chimeric primers during the primer extension are provided.