Trinity is a method for de novo assembly of full-length transcripts from RNA-Seq data without a reference genome. It efficiently reconstructs most transcripts, including alternatively spliced isoforms and transcripts from recently duplicated genes. Trinity outperforms other de novo transcriptome assemblers in recovering full-length transcripts across a broad range of expression levels, with sensitivity similar to methods that rely on genome alignments. It is particularly useful in the absence of a reference genome. Trinity consists of three software modules: Inchworm, Chrysalis, and Butterfly. Inchworm assembles reads into unique sequences of transcripts using a greedy k-mer-based approach. Chrysalis clusters related contigs and constructs de Bruijn graphs for each cluster. Butterfly analyzes the paths taken by reads and read pairings in the context of the de Bruijn graph to report all plausible transcript sequences, resolving alternatively spliced isoforms and transcripts derived from paralogous genes. Trinity was evaluated on data from fission yeast, mouse, and whitefly. In each case, it recovered most of the reference (annotated) expressed transcripts as full-length sequences, resolving alternative isoforms and duplicated genes. It performed better than other available transcriptome de novo assembly tools and similarly to methods relying on genome alignments. Trinity successfully reconstructed full-length transcripts in fission yeast and mouse, extending the 5' and 3' UTRs of genes and identifying novel transcribed sequences. It also identified long antisense transcripts and extended UTRs in fission yeast. In mouse, Trinity resolved splice isoforms and gene paralogs, extending the 5' and 3' UTRs of transcripts and identifying novel transcripts and exons. Trinity demonstrated high sequence fidelity, with a low base error rate. It outperformed other de novo assemblers in sensitivity and accuracy, particularly in capturing alternative splicing patterns and introns. It also performed well in reconstructing transcripts from the whitefly transcriptome, identifying allelic variants and alternatively spliced transcripts. Trinity is important for genome annotation and the study of non-model organisms. It provides a unified solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome. It is particularly useful for organisms with incomplete or missing reference genomes, such as the whitefly. Trinity's ability to capture multiple isoforms and its low base error rate make it crucial for maintaining acceptable levels of accuracy when characterizing genes.Trinity is a method for de novo assembly of full-length transcripts from RNA-Seq data without a reference genome. It efficiently reconstructs most transcripts, including alternatively spliced isoforms and transcripts from recently duplicated genes. Trinity outperforms other de novo transcriptome assemblers in recovering full-length transcripts across a broad range of expression levels, with sensitivity similar to methods that rely on genome alignments. It is particularly useful in the absence of a reference genome. Trinity consists of three software modules: Inchworm, Chrysalis, and Butterfly. Inchworm assembles reads into unique sequences of transcripts using a greedy k-mer-based approach. Chrysalis clusters related contigs and constructs de Bruijn graphs for each cluster. Butterfly analyzes the paths taken by reads and read pairings in the context of the de Bruijn graph to report all plausible transcript sequences, resolving alternatively spliced isoforms and transcripts derived from paralogous genes. Trinity was evaluated on data from fission yeast, mouse, and whitefly. In each case, it recovered most of the reference (annotated) expressed transcripts as full-length sequences, resolving alternative isoforms and duplicated genes. It performed better than other available transcriptome de novo assembly tools and similarly to methods relying on genome alignments. Trinity successfully reconstructed full-length transcripts in fission yeast and mouse, extending the 5' and 3' UTRs of genes and identifying novel transcribed sequences. It also identified long antisense transcripts and extended UTRs in fission yeast. In mouse, Trinity resolved splice isoforms and gene paralogs, extending the 5' and 3' UTRs of transcripts and identifying novel transcripts and exons. Trinity demonstrated high sequence fidelity, with a low base error rate. It outperformed other de novo assemblers in sensitivity and accuracy, particularly in capturing alternative splicing patterns and introns. It also performed well in reconstructing transcripts from the whitefly transcriptome, identifying allelic variants and alternatively spliced transcripts. Trinity is important for genome annotation and the study of non-model organisms. It provides a unified solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome. It is particularly useful for organisms with incomplete or missing reference genomes, such as the whitefly. Trinity's ability to capture multiple isoforms and its low base error rate make it crucial for maintaining acceptable levels of accuracy when characterizing genes.