Alternative Splicing, RNA Editing, and the Current Limits of Next Generation Sequencing

Abstract

The advent of next generation sequencing (NGS) has fostered a shift in basic analytic strategies of a gene expression analysis in diverse pathologies for the purposes of research, pharmacology, and personalized medicine. What was once highly focused research on individual signaling pathways or pathway members has, from the time of gene expression arrays, become a global analysis of gene expression that has aided in identifying novel pathway interactions, the discovery of new therapeutic targets, and the establishment of disease-associated profiles for assessing progression, stratification, or a therapeutic response. But there are significant caveats to this analysis that do not allow for the construction of the full picture. The lack of timely updates to publicly available databases and the “hit and miss” deposition of scientific data to these databases relegate a large amount of potentially important data to “garbage”, begging the question, “how much are we really missing?” This brief perspective aims to highlight some of the limitations that RNA binding/modifying proteins and RNA processing impose on our current usage of NGS technologies as relating to cancer and how not fully appreciating the limitations of current NGS technology may negatively affect therapeutic strategies in the long run.

RNA Splicing in Cancer and Targeted Therapies

“…RNA editing is an important type of post-transcriptional processing, which changes the primary RNA sequence through the insertion/deletion or modification of specific nucleotides. Piazzi et al, in their review [4], highlighted RNA editing and its link with pathophysiology. The most common modification in humans is deamination.…”

“…However, there are significant caveats to these analyses, which are crucial roadblocks against our progress in obtaining the complete picture. Piazzi et al, in their review [4], discussed this issue and highlighted several problems. One of the biggest issues in interpreting RNA-seq data is the absence of many alternatively spliced transcript isoforms in the databanks.…”

How Error Correction Affects PCR Deduplication: A Survey Based on UMI Datasets of Short Reads

“…Next-Generation Sequencing (NGS) has revolutionized genomics research, enabling extensive acquisition of genome-wide data with unprecedented speed, precision, and cost-effectiveness and making significant progress in the field of DNA sequencing (DNA-Seq) [1] and RNA sequencing (RNA-seq) [2]. NGS data plays a vital role in various downstream analyses, including estimation of microbial diversity [3], variant calling [4], immune cell responses [5, 6], RNA quantification [7], cancer mutation detection [8], de novo genome assembly [9], de novo transcriptome assembly [10] and nonclinical genotoxicity and carcinogenicity testing [11], detecting allele-specific expression [12], isomiR identification [13] and genome base editing [14]. One of the key steps in NGS is the polymerase chain reaction (PCR) process which is purposely used in the library construction and cluster amplification to increase the number of DNA/RNA molecule fragments.…”