Journal: bioRxiv

Article Title: Comprehensive Assessment of Isoform Detection Methods for Third-Generation Sequencing Data

doi: 10.1101/2023.08.03.551905

Figure Lengend Snippet: For the preparation of simulated datasets, YASIM was utilized to simulate TGS RNA-seq datasets with variations in sequencing depth, transcriptome complexity index, sequencing read completeness, sequencing error rates, and completeness of the reference annotation. Experimental datasets were obtained from publicly available TGS RNA-seq datasets of four species, including human, mouse, drosophila, and C. elegans. Additionally, in-house TGS RNA-seq datasets were generated from human embryonic stem cells in both Naïve and Primed conditions. The performance of the software was assessed from multiple perspectives, including accuracy, classification of identified isoforms, pairwise similarity between the results, and downstream analysis of differential isoform usage (DIU). Furthermore, computational resource consumption by each method was analyzed.

Article Snippet: Naïve and primed hESCs (H1 cell line, Wicell Research Institute, Inc., WA01-pcbc) were maintained following previously described protocols .

Techniques: RNA Sequencing Assay, Sequencing, Generated, Software

Journal: bioRxiv

Article Title: Comprehensive Assessment of Isoform Detection Methods for Third-Generation Sequencing Data

doi: 10.1101/2023.08.03.551905

Figure Lengend Snippet: A, B. Five different isoform types and the total counts of isoform events detected by nine different methods across 25 experimental datasets collected using the Nanopore (A) or Pacbio platform (B). FSM, ISM, NIC, NNC represent full splice match, incomplete splice match, novel in catalog, and novel not in catalog, respectively. The size of each sector is proportional to the largest count of isoform events detected by the nine different methods for each dataset. The results for the nine methods were hierarchically clustered. The publicly available experimental datasets originate from the following sources: C1: L1 larval stage of C. elegans, C2: mix stage of C. elegans, C3: young adult stage of C. elegans; C4: Wildtype C. elegans total RNA replicate 1, C5: Wildtype C. elegans total RNA replicate 2, D1: Drosophila, D2: Drosophila testis, M1: mouse activated CD8 T cell, M2: mouse naïve CD8 T cell, M3: mouse retinal cells (control), M4: mouse retinal cells (glaucomatous), M5: mouse CD4SP cells, M6: mouse CD8SP cells, M7: mouse neural stem cells (E15.5), M8: mouse neural stem cells (P1.5), M9: mouse cerebral cells, M10: mouse hippocampus cells. H1: human Beta cells, H2: human Beta cells treated with cytokines, H3: human Hela cells, H4: human iPSC cells. The TGS RNA-seq dataset on Naïve and Primed hESCs was generated in this study.

Article Snippet: Naïve and primed hESCs (H1 cell line, Wicell Research Institute, Inc., WA01-pcbc) were maintained following previously described protocols .

Techniques: Control, RNA Sequencing Assay, Generated

Journal: bioRxiv

Article Title: Comprehensive Assessment of Isoform Detection Methods for Third-Generation Sequencing Data

doi: 10.1101/2023.08.03.551905

Figure Lengend Snippet: A, B. Heatmap showing pairwise Jaccard statistics representing the overlap of isoforms identified by nine different methods across 25 experimental datasets collected using the Nanopore (A) or Pacbio platform (B). The * symbol denotes the top three Jaccard overlaps in each dataset. The publicly available experimental datasets originate from the following sources: C1: L1 larval stage of C. elegans, C2: mix stage of C. elegans, C3: young adult stage of C. elegans; C4: Wildtype C. elegans total RNA replicate 1, C5: Wildtype C. elegans total RNA replicate 2, D1: Drosophila, D2: Drosophila testis, M1: mouse activated CD8 T cell, M2: mouse naïve CD8 T cell, M3: mouse retinal cells (control), M4: mouse retinal cells (glaucomatous), M5: mouse CD4SP cells, M6: mouse CD8SP cells, M7: mouse neural stem cells (E15.5), M8: mouse neural stem cells (P1.5), M9: mouse cerebral cells, M10: mouse hippocam-pus cells. H1: human Beta cells, H2: human Beta cells treated with cytokines, H3: human Hela cells, H4: human iPSC cells. The TGS RNA-seq dataset on Naïve and Primed hESCs was generated in this study.

Article Snippet: Naïve and primed hESCs (H1 cell line, Wicell Research Institute, Inc., WA01-pcbc) were maintained following previously described protocols .

Techniques: Control, RNA Sequencing Assay, Generated

Journal: bioRxiv

Article Title: Comprehensive Assessment of Isoform Detection Methods for Third-Generation Sequencing Data

doi: 10.1101/2023.08.03.551905

Figure Lengend Snippet: A. Bar plot showing the DIU calling accuracy using simulated data with nine different methods. B. Bar plot showing alternative isoform types for up-regulated and down-regulated DIU in Primed hESC compared with Naïve hESCs detected using nine methods. The analysis includes TGS RNA-seq dataset from Naïve and Primed hESC generated in this study, as well as NGS RNA-seq dataset from Naïve and Primed hESC. A3: alternative 3’ splice site, A5: alternative 5’ splice site, ATSS: alternative transcript start site, ATTS: alternative transcript terminated site, ES: exon skipping, IR: intron retention, MEE: mutually exclusive exon, MES: mutually exclusive splicing. C. Bar plot showing the number of up-regulated and down-regulated DIU isoforms in Primed hESC with different switching consequences. NMD: nonsense-mediated decay; ORF: open reading frame. D, E. UpSet plot showing the number of overlapped DIU genes up-regulated (D) and down-regulated (E) in Primed hESC compared with Naïve hESCs identified by different methods and from the NGS data. F. IGV screenshot displaying TGS RNA-seq coverages and splicing junctions of RPL39L gene isoforms in Naïve and Primed hESC. The gene model for different transcript isoforms of RPL39L is shown under the tracks. RPL39L-L and RPL39L-S represent isoforms on the gene reference, and RPL39L-UN represents the novel isoform detected in this study. Red arrows indicate primers used in RT-qPCR validation experiments. G. Bar plot representing the TGS read proportions of the three isoforms of RPL39L in Naïve and Primed hESC. H. Bar plot representing the RT-qPCR results of the three different isoforms of RPL39L . The RT-qPCR was performed using the isoform-specific primers shown in F. Each group involves three biological replicates, and each bar represents the fold change relative to the expression of RPL39L-S in Primed hESC. Error bars represent standard deviation. Statisti-cal analysis was performed with a two-sided T-test (* P < 0.05).

Article Snippet: Naïve and primed hESCs (H1 cell line, Wicell Research Institute, Inc., WA01-pcbc) were maintained following previously described protocols .

Techniques: RNA Sequencing Assay, Generated, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Cells

Article Title: Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients

doi: 10.3390/cells10113099

Figure Lengend Snippet: The process of reprogramming of Turner syndrome PBMCs into hiPSCs and characterization of hiPSCs-TS and colonies. ( A ) Schematic protocol used to reprogramming PBMCs into hiPSCs. ( B ) PBMCs with ten days of expansion in a specific medium for enrichment erythroblastic populations; the emergence of the first hiPSCs-TS colonies maintained in hESCs medium on MEF. ( C ) Characterization of hiPSCs-TS of Pt.1, Pt.2, Pt.3, Pt.4 and Pt.5. Phase contrast image showed hiPSCs maintained in mTeSR-1 medium on Geltrex; positively stain of alkaline phosphatase; Immunofluorescence assay showed the presence of pluripotency markers OCT4, SOX2, NANOG. Nuclei were stained with Hoechst (blue). Scale bars s: 200 μm and 100 μm. The data was compiled from at least three technical replicates. ( D ) Quantification of the relative expression of OCT4 , SOX2 , NANOG , KLF4 , and c-MYC genes in all hiPSCs lines and subclones (p15). Individual quantifications by qRT-PCR were normalized using the Β-ACTIN gene. The ESCs-H1 line was used as a positive control and was not considered in the statistical model. All qRT-PCR from hiPSCs-TS and control data were compiled from at least three biological and technical replicates. p values were calculated by Two-way ANOVA, followed by Tukey’s multiple comparisons test as appropriate (letters a-b-c show difference between groups). Erros bars denote represent SD.

Article Snippet: The human embryonic stem cell (hESCs) line H1 (cat.# WA01-cGMP Material) was bought from WiCell International Stem Cell Bank, and was first published by Thomson et al. [ ].

Techniques: Staining, Immunofluorescence, Expressing, Quantitative RT-PCR, Positive Control

Journal: Cells

Article Title: Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients

doi: 10.3390/cells10113099

Figure Lengend Snippet: Cytogenetic analysis of all lines of hiPSCs-TS and controls. ( A – C ) phase contrast and karyotype of hESCs-H1 (p30) and hiPSCs male and female control (p20). 2). ( D – G ) phase contrast and karyotype of hiPSCs TS (p20). 3). ( H ) Phase contrast and karyotype of hiPSCs TS–Pt.2 (p20). Cytogenetic analyses revealed reciprocal chromosome translocations between chromosomes 11 with breakage at the q arm, and chromosome 12 with a breakpoint at the p arm (der(11;12)(q10;10), der(11;12)(p10;p10)). Scale bars indicate 400 μm.

Article Snippet: The human embryonic stem cell (hESCs) line H1 (cat.# WA01-cGMP Material) was bought from WiCell International Stem Cell Bank, and was first published by Thomson et al. [ ].

Techniques: