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RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been developed. However, the relative advantage of each method for degraded RNA and low-input RNA, such as RNA samples collected in the field of clinical setting, has remained unknown. The Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range, and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared the correlation, the number of detected expressing genes, and the expression levels with the Standard RNA-Seq method. Although the performance of RamDA-Seq was similar to that of Standard RNA-Seq, the performance for low-input RNA and degraded RNA has decreased. The performance of SMART-Seq was better than xGen and RamDA-Seq in low-input RNA and degraded RNA. Furthermore, the depletion of ribosomal RNA (rRNA) improved the performance of SMART-Seq and xGen due to increased expression levels. SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low-input and degraded RNA.

Background mRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis. Results We observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated. Conclusions TruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime with regards to the detected number of transcripts and splicing events among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.

Human hereditary malformation syndromes are caused by mutations in the genes of the signal transduction molecules involved in fetal development. Among them, the Sonic hedgehog (SHH) signaling pathway is the most important, and many syndromes result from its disruption. In this review, we summarize the molecular mechanisms and role in embryonic morphogenesis of the SHH pathway, then classify the phenotype of each malformation syndrome associated with mutations of major molecules in the pathway. The output of the SHH pathway is shown as GLI activity, which is generated by SHH in a concentration-dependent manner, i.e., the sum of activating form of GLI (GLIA) and repressive form of GLI (GLIR). Which gene is mutated and whether the mutation is loss-of-function or gain-of-function determine in which concentration range of SHH the imbalance occurs. In human malformation syndromes, too much or too little GLI activity produces symmetric phenotypes affecting brain size, craniofacial (midface) dysmorphism, and orientation of polydactyly with respect to the axis of the limb. The symptoms of each syndrome can be explained by the GLIA/R balance model.

The full-length double-strand cDNA sequencing, one of the RNA-Seq methods, is a powerful method used to investigate the transcriptome status of a gene of interest, such as its transcription level and alternative splicing variants. Furthermore, full-length double-strand cDNA sequencing has the advantage that it can create a library from a small amount of sample and the library can be applied to long-read sequencers in addition to short-read sequencers. Nevertheless, one of our previous studies indicated that the full-length double-strand cDNA sequencing yields non-specific genomic DNA amplification, affecting transcriptome analysis, such as transcript quantification and alternative splicing analysis. In this study, it was confirmed that it is possible to produce the RNA-Seq library from only genomic DNA and that the full-length double-strand cDNA sequencing of genomic DNA yielded non-specific genomic DNA amplification. To avoid non-specific genomic DNA amplification, two methods were examined, which are the DNase I-treated full-length double-strand cDNA sequencing and poly(A) capture full-length double-strand cDNA sequencing. Contrary to expectations, the non-specific genomic DNA amplification was increased and the number of the detected expressing genes was reduced in DNase I-treated full-length double-strand cDNA sequencing. On the other hand, in the poly(A) capture full-length double-strand cDNA sequencing, the non-specific genomic DNA amplification was significantly reduced, accordingly the accuracy and the number of detected expressing genes and splicing events were increased. The expression pattern and percentage spliced in index of splicing events were highly correlated. Our results indicate that the poly(A) capture full-length double-strand cDNA sequencing improves transcript quantification accuracy and the detection ability of alternative splicing events. It is also expected to contribute to the determination of the significance of DNA variants to splicing events.

Cancer gene panel testing requires accurate detection of somatic mosaic mutations, as the test sample consists of a mixture of cancer cells and normal cells; each minor clone in the tumor also has different somatic mutations. Several studies have shown that the different types of software used for variant calling for next generation sequencing (NGS) can detect low-frequency somatic mutations. However, the accuracy of these somatic variant callers is unknown. We performed cancer gene panel testing in duplicate experiments using three different high-fidelity DNA polymerases in pre-capture amplification steps and analyzed by three different variant callers, Strelka2, Mutect2, and LoFreq. We selected six somatic variants that were detected in both experiments with more than two polymerases and by at least one variant caller. Among them, five single nucleotide variants were verified by CEL nuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) and Sanger sequencing. In silico analysis indicated that the FBXW7 and MAP3K1 missense mutations cause damage at the protein level. Comparing three somatic variant callers, we found that Strelka2 detected more variants than Mutect2 and LoFreq. We conclude that dual sequencing with Strelka2 analysis is useful for detection of accurate somatic mutations in cancer gene panel testing.

Here, we report a novel PROS1 splicing mutation in a patient with type I protein S deficiency. Qualitative and quantitative analysis of pathogenic splicing variants at the mRNA level was performed by long-range PCR-based targeted DNA and RNA sequencing. A base substitution in the exon 4 splicing donor site activates a potential splicing donor site in intron 4, resulting in an in-frame insertion of 48 bases (16 amino acids).

Recessive dystrophic epidermolysis bullosa is a genetic collagen disorder characterized by skin fragility that leads to generalized severe blistering, wounds, and scarring. In this report, we present a patient with a novel COL7A1 homozygous nonsense variant, c.793C>T p.(Gln265*). Although the parents were not consanguineous, both were heterozygous carriers of the variant. Single nucleotide polymorphism (SNP) array analysis revealed an isodisomy area on 3p22.1p21.1, encompassing COL7A1, suggesting that the variant originated from a common ancestor.

Dendritic cells (DCs) are the most potent antigen-presenting cells, playing an essential role in the pathogen and tumor recognition, and anti-tumor immunity, and linking both the innate and adaptive immunity. The monocyte-derived DCs generated by ex vivo culture, have been used for cancer immunotherapy to eliminate tumor; however, the clinical efficacies are not sufficient, and further improvement is essential. In this study, we established a method to generate DCs using small molecule compounds for cancer immunotherapy. We observed an increase in the percentage of CD11c + I-A/I-E high cells, representing DCs, by adding four small molecular inhibitors: Y27632, PD0325901, PD173074, and PD98059 (abbreviated as YPPP), in mouse bone marrow (BM) culture with granulocyte-macrophage colony stimulating factor (GM-CSF). BM-derived DCs cultured with YPPP (YPPP-DCs) showed high responsiveness to lipopolysaccharide stimulation, resulting in increased interleukin (IL) -12 production and enhanced proliferation activity when co-cultured with naïve T cells compared with the vehicle control. RNA-seq analysis revealed an upregulation of peroxisome proliferator - activated receptor (PPAR) γ associated genes increased in YPPP-DCs. In tumor models treated with anti-programmed death (PD) -1 therapies, mice injected intratumorally with YPPP-DCs as a DCs vaccine exhibited reduced tumor growth and increased survival. These findings suggested that our method would be useful for the induction of DCs that efficiently activate effector T cells for cancer immunotherapy.

Ensuring cooperation among formerly autonomous cells has been a central challenge in the evolution of multicellular organisms. One solution is monoclonality, but this option still leaves room for exploitative behavior, as it does not eliminate genetic and epigenetic variability. We therefore hypothesized that embryonic development must be protected by robust regulatory mechanisms that prevent aberrant clones from superseding wild-type cells. Using a genome-wide screen in murine induced pluripotent stem cells, we identified a network of genes (centered on p53, topoisomerase 1, and olfactory receptors) whose down-regulation caused the cells to replace wild-type cells in vitro and in the mouse embryo—without perturbing normal development. These genes thus appear to fulfill an unexpected role in fostering cell cooperation.

There are two combinations of heterozygous mutation, i.e., in trans, which carries mutations on different alleles, and in cis, which carries mutations on the same allele. Because only in trans compound heterozygous mutations have been implicated in autosomal recessive diseases, it is important to distinguish them for clinical diagnosis. However, conventional phase analysis is limited because of the large target size of genomic DNA. Here, we performed a genetic analysis on a patient with Wilson disease, and we detected two heterozygous mutations chr13:51958362;G>GG (NM_000053.4:c.2304dup r.2304dup p.Met769HisfsTer26) and chr13:51964900;C>T (NM_000053.4:c.1841G>A r.1841g>a p.Gly614Asp) in the causative gene ATP7B. The distance between the two mutations was 6.5 kb in genomic DNA but 464 bp in mRNA. Targeted double-stranded cDNA sequencing-based phase analysis was performed using direct adapter ligation library preparation and paired-end sequencing, and we elucidated they are in trans compound heterozygous mutations. Trio analysis showed that the mutation (chr13:51964900;C>T) derived from the father and the other mutation from the mother, validating that the mutations are in trans composition. Furthermore, targeted double-stranded cDNA sequencing-based phase analysis detected the differential allelic expression, suggesting that the mutation (chr13:51958362;G>GG) caused downregulation of expression by nonsense-mediated mRNA decay. Our results indicate that targeted double-stranded cDNA sequencing-based phase analysis is useful for determining compound heterozygous mutations and confers information on allelic expression.