Trinity is a novel method for de novo reconstruction of full-length transcripts from RNA-Seq data without a reference genome. It consists of three modules: Inchworm, Chrysalis, and Butterfly. Inchworm assembles reads into unique transcript sequences, Chrysalis constructs de Bruijn graphs for clusters of related contigs, and Butterfly resolves alternative splice forms and paralogous transcripts. Trinity outperforms other de novo transcriptome assembly tools and is comparable to methods relying on genome alignments. It successfully reconstructs most transcripts in samples from fission yeast, mouse, and whitefly, including alternative splice isoforms and transcripts from recently duplicated genes. Trinity also extends 5' and 3' UTRs and identifies novel transcribed sequences and long antisense transcripts. It achieves high sequence fidelity, with a low error rate, and accurately resolves splice isoforms and gene paralogs. Trinity is particularly effective in organisms without a sequenced genome, such as the whitefly. It provides a unified and general solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome. Trinity is publicly available and has been validated on multiple species, demonstrating its robustness and accuracy in transcriptome assembly.Trinity is a novel method for de novo reconstruction of full-length transcripts from RNA-Seq data without a reference genome. It consists of three modules: Inchworm, Chrysalis, and Butterfly. Inchworm assembles reads into unique transcript sequences, Chrysalis constructs de Bruijn graphs for clusters of related contigs, and Butterfly resolves alternative splice forms and paralogous transcripts. Trinity outperforms other de novo transcriptome assembly tools and is comparable to methods relying on genome alignments. It successfully reconstructs most transcripts in samples from fission yeast, mouse, and whitefly, including alternative splice isoforms and transcripts from recently duplicated genes. Trinity also extends 5' and 3' UTRs and identifies novel transcribed sequences and long antisense transcripts. It achieves high sequence fidelity, with a low error rate, and accurately resolves splice isoforms and gene paralogs. Trinity is particularly effective in organisms without a sequenced genome, such as the whitefly. It provides a unified and general solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome. Trinity is publicly available and has been validated on multiple species, demonstrating its robustness and accuracy in transcriptome assembly.