wa01-pcbc  (WiCell Research Institute Inc)

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