The human genome contains more than 200,000 gene isoforms. However, different isoforms can be highly similar, and with an average length of 1.5kb remain difficult to study with short read sequencing. To systematically evaluate the ability to study the transcriptome at a resolution of individual isoforms we profiled 5 human cell lines with short read cDNA sequencing and Nanopore long read direct RNA, amplification-free direct cDNA, PCR-cDNA sequencing. The long read protocols showed a high level of consistency, with amplification-free RNA and cDNA sequencing being most similar. While short and long reads generated comparable gene expression estimates, they differed substantially for individual isoforms. We find that increased read length improves read-to-transcript assignment, identifies interactions between alternative promoters and splicing, enables the discovery of novel transcripts from repetitive regions, facilitates the quantification of full-length fusion isoforms and enables the simultaneous profiling of m6A RNA modifications when RNA is sequenced directly. Our study demonstrates the advantage of long read RNA sequencing and provides a comprehensive resource that will enable the development and benchmarking of computational methods for profiling complex transcriptional events at isoform-level resolution.

Therapeutic strategies against KRAS mutant colorectal cancers are developed using cell line models, which do not accurately represent the transcriptome driven by oncogenic KRAS in tumors. We sought to identify a KRAS-associated gene signature from colorectal tumors to develop a precise treatment strategy. Integrative analysis of quantitative KRAS mutation detection and matched gene expression profiling in 55 CRC bulk tumors was carried out to define a gene signature enriched in CRC tumors with high KRAS mutation. The KRAS-associated gene signature identified exhibits functional enrichment in cell cycle and mitosis processes, and includes mitotic transcription factor, FOXM1. Combination treatment of CDK4/6 inhibitor Palbociclib and MEK inhibitor PD0325901 was tested in KRAS-mutant, BRAF-mutant CRC, normal colon epithelial lines and xenografts models to determine their efficacy and toxicity and to monitor the changes in the gene signature. Inhibiting CDK4/6, an upstream regulator of FOXM1, and MEK synergistically depleted FOXM1 and KRAS-associated gene signature, suggesting that CDK4/6 and MEK regulate the KRAS gene signature. The combined inhibition of CDK4/6 and MEK elicited a robust therapeutic response in KRAS-dependent and BRAF-mutant CRC, both in vitro and in vivo and this correlated with downregulation of the KRAS-associated gene signature. Our preclinical study demonstrated the efficacy of Palbociclib and PD0325901 combinatorial treatment selectively in KRAS-dependent and BRAF-mutant CRC but not in normal colon epithelial cells. The KRAS-associated gene signature could facilitate the identification of responsive metastatic CRC to this therapeutic strategy in clinical settings.

The small yellow croaker (Larimichthys polyactis) is one of the most commercially exploited marine fishes along the coast of the Yellow-Bohai Sea and the East China Sea. In this study, we used next-generation high-throughput 2b-RAD-seq technology to identify novel SNPs in L. polyactis. We scored a total of 1 374 008 putative SNPs genome-wide. Further filtration yielded a final dataset of 6457 high-quality SNPs. These SNP markers presented sufficient power in detecting genetic distinction between two wild samples from the Yellow-Bohai Sea and one captive sample from the East China Sea, and inbreeding reflected by strong heterozygote deficiency within the samples; some of the genetic polymorphisms are diagnostic among the samples and related to biological functions. The panel of SNPs could be used as powerful tools in further population genetic and stock assessment research in L. polyactis as it has relatively scarce genomic resources. The findings from this study will advance our understanding of population and functional genomics for facilitating fishery resource management and developing desirable characteristics for the benefit of culture and farming of L. polyactis.