Reassessment of Marker Genes in Human Induced Pluripotent Stem Cells for Enhanced Quality Control

Author: Jochen Dobner E-Mail: [e-mail] Description: This data repository contains binary alignment map (BAM) files derived from Oxford Nanopore Technologies (ONT) long-read transcriptome sequencing of human induced pluripotent stem cells (iPSCs). Human iPSC12 (female) cells were differentiated into each of the three primary germ layers endoderm, ectoderm, and mesoderm by directed differentiation in biological duplicates. Total RNA was extracted, double stranded cDNA reverse transcribed, amplified and subjected to sequencing library preparation (Sequencing by Ligation Native Barcoding). Samples were sequenced on a PromethION flow cell on a P2 Solo device. Raw FAST5 files were base-called using guppy basecaller (v6.4.6) and aligned to the human reference genome (GRCh38) using minimap2 (v2.24). Transcript counting and differential gene expression analysis were performed using R (v4.2.2) packages Rsubread (v2.12.3) and edgeR (v3.40.2). It was chosen to upload reference-sorted BAM instead of FASTQ files to this repository, because ever growing data masses will likely become a bottleneck in the future. BAM files are smaller in size compared to FASTQ, but contain all their information additional to the alignment. BAM files can be reconverted to FASTQ files, e.g. by the SAMtools command 'samtools bam2fq INPUT.bam > OUTPUT.fastq. The barcodes refer to the following samples: barcode01: iPSC12 Undifferentiated n1 barcode02: iPSC12 Endoderm n1 barcode03: iPSC12 Ectoderm n1 barcode04: iPSC12 Mesoderm n1 barcode05: iPSC12 Undifferentiated n2 barcode06: iPSC12 Endoderm n2 barcode07: iPSC12 Mesoderm n2 barcode08: iPSC12 Ectoderm n2 In an updated version (2024-05-16) of this Dataset, additional ONT long-read transcriptome sequencing data in the CRAM format (it is recommended to convert to BAM prior analysis) of intermediate trilineage-differentiated iPSC12 cells was added (ts_cram.zip). This BAM files are derived from shallow sequencing of Undifferentiated (P0), Endoderm D2 (E2) and D3 (E3), Mesoderm D2 (M2) and D3 (M3), and Ectoderm D2 (Ec2), D3 (Ec3) and D5 (Ec5) and has been prepared according to the description above. The title of this repository refers to the manuscript of the same title which is submitted for publication. More details can be found once the manuscript is published. For further details, please contact the repository owner.