RNA sequencing has been widely applied for gene isoform quantification, but limitations exist in quantifying isoforms of complex genes accurately, especially for short reads. Here we identify genes that are difficult to quantify accurately with short reads and illustrate the information benefit of using long reads to quantify these regions. We present miniQuant, which ranks genes with quantification errors caused by the ambiguity of read alignments and integrates the complementary strengths of long reads and short reads with optimal combination in a gene- and data-specific manner to achieve more accurate quantification. These results are supported by rigorous mathematical proofs, validated with a wide range of simulation data, experimental validations and more than 17,000 public datasets from GTEx, TCGA and ENCODE consortia. We demonstrate miniQuant can uncover isoform switches during the differentiation of human embryonic stem cells to pharyngeal endoderm and primordial germ cell-like cells.