Evaluation and application of RNA-Seq by MinION.

The current RNA-Seq method analyses fragments of mRNAs, from which it is occasionally difficult to reconstruct the entire transcript structure. Here, we performed and evaluated the recent procedure for full-length cDNA sequencing using the Nanopore sequencer MinION. We applied MinION RNA-Seq for various applications, which would not always be easy using the usual RNA-Seq by Illumina. First, we examined and found that even though the sequencing accuracy was still limited to 92.3%, practically useful RNA-Seq analysis is possible. Particularly, taking advantage of the long-read nature of MinION, we demonstrate the identification of splicing patterns and their combinations as a form of full-length cDNAs without losing precise information concerning their expression levels. Transcripts of fusion genes in cancer cells can also be identified and characterized. Furthermore, the full-length cDNA information can be used for phasing of the SNPs detected by WES on the transcripts, providing essential information to identify allele-specific transcriptional events. We constructed a catalogue of full-length cDNAs in seven major organs for two particular individuals and identified allele-specific transcription and splicing. Finally, we demonstrate that single-cell sequencing is also possible. RNA-Seq on the MinION platform should provide a novel approach that is complementary to the current RNA-Seq.

Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer.

Here, we employed cDNA amplicon sequencing using a long-read portable sequencer, MinION, to characterize various types of mutations in cancer-related genes, namely, EGFR, KRAS, NRAS and NF1. For homozygous SNVs, the precision and recall rates were 87.5% and 91.3%, respectively. For previously reported hotspot mutations, the precision and recall rates reached 100%. The precise junctions of EML4-ALK, CCDC6-RET and five other gene fusions were also detected. Taking advantages of long-read sequencing, we conducted phasing of EGFR mutations and elucidated the mutational allelic backgrounds of anti-tumor drug-sensitive and resistant mutations, which could provide useful information for selecting therapeutic approaches. In the H1975 cells, 72% of the reads harbored both L858R and T790M mutations, and 22% of the reads harbored neither mutation. To ensure that the clinical requirements can be met in potentially low cancer cell populations, we further conducted a serial dilution analysis of the template for EGFR mutations. Several percent of the mutant alleles could be detected depending on the yield and quality of the sequencing data. Finally, we characterized the mutation genotypes in eight clinical samples. This method could be a convenient long-read sequencing-based analytical approach and thus may change the current approaches used for cancer genome sequencing.

Characterization of STAT6 target genes in human B cells and lung epithelial cells.

Using ChIP Seq, we identified 556 and 467 putative STAT6 target sites in the Burkitt's lymphoma cell line Ramos and in the normal lung epithelial cell line BEAS2B, respectively. We also examined the positions and expression of transcriptional start sites (TSSs) in these cells using our TSS Seq method. We observed that 44 and 132 genes in Ramos and BEAS2B, respectively, had STAT6 binding sites in proximal regions of their previously reported TSSs that were up-regulated at the transcriptional level. In addition, 406 and 109 of the STAT6 target sites in Ramos and BEAS2B, respectively, were located in proximal regions of previously uncharacterized TSSs. The target genes identified in Ramos and BEAS2B cells in this study and in Th2 cells in previous studies rarely overlapped and differed in their identity. Interestingly, ChIP Seq analyses of histone modifications and RNA polymerase II revealed that chromatin formed an active structure in regions surrounding the STAT6 binding sites; this event also frequently occurred in different cell types, although neither STAT6 binding nor TSS induction was observed. The rough landscape of STAT6-responsive sites was found to be shaped by chromatin structure, but distinct cellular responses were mainly mediated by distinct sets of transcription factors.

Massive transcriptional start site analysis of human genes in hypoxia cells.

Combining our full-length cDNA method and the massively parallel sequencing technology, we developed a simple method to collect precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high throughput manner. We applied this method to observe gene-expression changes in a colon cancer cell line cultured in normoxic and hypoxic conditions. We generated more than 100 million 36-base TSS-tag sequences and revealed comprehensive features of hypoxia responsive alterations in the transcriptional landscape of the human genome. The features include presence of inducible 'hot regions' in 54 genomic regions, 220 novel hypoxia inducible promoters that may drive non-protein-coding transcripts, 191 hypoxia responsive alternative promoters and detailed views of 120 novel as well as known hypoxia responsive genes. We further analyzed hypoxic response of different cells using additional 60 million TSS-tags and found that the degree of the gene-expression changes were different among cell lines, possibly reflecting cellular robustness against hypoxia. The novel dynamic figure of the human gene transcriptome will deepen our understanding of the transcriptional program of the human genome as well as bringing new insights into the biology of cancer cells in hypoxia.