Characterization of the human ESC transcriptome by hybrid sequencing

Abstract

Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, fulllength mRNA isoforms are not captured. On the other hand, thirdgeneration sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete.isoform discovery | PacBio | hESC transcriptome | alternative splicing | lncNRA

The dynamic landscape of fission yeast meiosis alternative-splice isoforms

“…PacBio sequencing features very long reads, which are suitable for full-length cDNA sequencing. Recently, PacBio sequencing has been used to examine the transcriptome in multiple metazoan organisms, such as chicken and human, and multiple plants, and identified thousands of annotated and novel isoforms (Au et al 2013;Sharon et al 2013;Martin et al 2014;Thomas et al 2014;Tilgner et al 2014;Dong et al 2015;Minoche et al 2015). To systematically characterize isoforms and explore their differential regulations in fission yeast, we performed time-course isoform-level profiling during the meiosis of S. pombe.…”

Thymus cDNA library survey uncovers novel features of immune molecules in Chinese giant salamander Andrias davidianus

“…Expressed sequence tags (ESTs) strategy based on cDNA library was employed, because it has provided a powerful platform for gene discovery, especially in organisms that have little genomic information (Giallourakis et al, 2013). Although conventional Sanger sequencing is time consuming, it yields high-quality and long reads, which is reliable for the assembly of gene isoforms (Au et al, 2013), and is therefore still used as an effective strategy in the next-generation sequencing era.…”

Transcriptome Survey of a Marine Food Fish: Asian Seabass (Lates calcarifer)

“…However, a number of these assemblies, including ours, have resulted in a high number of fragmented contigs [41][42][43]. As one of the vital factors for achieving a good assembly is the read length, the advent of long-read technologies such as Pacific Biosciences' Isoform Sequencing could help in improving the contiguity of de novo transcriptome assemblies [44,45].…”