RT Journal
A1 Monzó, Carolina
A1 Frankish, Adam
A1 Conesa, Ana
T1 Notable challenges posed by long-read sequencing for the study of transcriptional diversity and genome annotation
JF Genome Research 
JO Genome Research 
YR 2025 
FD April 01 
VO 35 
IS 4 
SP 583 
OP 592 
DO 10.1101/gr.279865.124 
UL http://genome.cshlp.org/content/35/4/583.abstract 
AB Long-read sequencing (LRS) technologies have revolutionized transcriptomic research by enabling the comprehensive sequencing of full-length transcripts. Using these technologies, researchers have reported tens of thousands of novel transcripts, even in well-annotated genomes, while developing new algorithms and experimental approaches to handle the noisy data. The Long-read RNA-seq Genome Annotation Assessment Project community effort benchmarked LRS methods in transcriptomics and validated many novel, lowly expressed, often times sample-specific transcripts identified by long reads. These molecules represent deviations of the major transcriptional program that were overlooked by short-read sequencing methods but are now captured by the full-length, single-molecule approach. This Perspective discusses the challenges and opportunities associated with LRS’ capacity to unravel this fraction of the transcriptome, in terms of both transcriptome biology and genome annotation. For transcriptome biology, we need to develop novel experimental and computational methods to effectively differentiate technology errors from rare but real molecules. For genome annotation, we must agree on the strategy to capture molecular variability while still defining reference annotations that are useful for the genomics community.