RT Journal
A1 Nudelman, German
A1 Frasca, Antonio
A1 Kent, Brandon
A1 Sadler, Kirsten C.
A1 Sealfon, Stuart C.
A1 Walsh, Martin J.
A1 Zaslavsky, Elena
T1 High resolution annotation of zebrafish transcriptome using long-read sequencing
JF Genome Research 
JO Genome Research 
YR 2018 
FD September 01 
VO 28 
IS 9 
SP 1415 
OP 1425 
DO 10.1101/gr.223586.117 
UL http://genome.cshlp.org/content/28/9/1415.abstract 
AB With the emergence of zebrafish as an important model organism, a concerted effort has been made to study its transcriptome. This effort is limited, however, by gaps in zebrafish annotation, which are especially pronounced concerning transcripts dynamically expressed during zygotic genome activation (ZGA). To date, short-read sequencing has been the principal technology for zebrafish transcriptome annotation. In part because these sequence reads are too short for assembly methods to resolve the full complexity of the transcriptome, the current annotation is rudimentary. By providing direct observation of full-length transcripts, recently refined long-read sequencing platforms can dramatically improve annotation coverage and accuracy. Here, we leveraged the SMRT platform to study the transcriptome of zebrafish embryos before and after ZGA. Our analysis revealed additional novelty and complexity in the zebrafish transcriptome, identifying 2539 high-confidence novel transcripts that originated from previously unannotated loci and 1835 high-confidence new isoforms in previously annotated genes. We validated these findings using a suite of computational approaches including structural prediction, sequence homology, and functional conservation analyses, as well as by confirmatory transcript quantification with short-read sequencing data. Our analyses provided insight into new homologs and paralogs of functionally important proteins and noncoding RNAs, isoform switching occurrences, and different classes of novel splicing events. Several novel isoforms representing distinct splicing events were validated through PCR experiments, including the discovery and validation of a novel 8-kb transcript spanning multiple mir-430 elements, an important driver of early development. Our study provides a significantly improved zebrafish transcriptome annotation resource.