Genner, R. | bioRxiv Science

bioRxiv2026

The complete genome of the KOLF2.1J reference iPSC line

While induced pluripotent stem cells (iPSCs) have gained popularity in studying neurodegenerative diseases, the heterogeneity of stem cells used across studies impacts cross-study comparison. The iPSC Neurodegenerative Disease Initiative (iNDI) selected the KOLF2.1J cell line and prioritized its use as a reference standard for studying the effects of pathogenic variants on cell biology due to its stability and neutral neurodegenerative disease genetic risk. This cell line, and its derivatives expressing over 100 variants related to Alzheimers disease, Parkinsons disease, and other neurological diseases, are available for academic and industry access. Current genomic data analyses are limited by the use of a human reference genome that does not capture the complete genetic background of a given iPSC line. While in the future this issue may be partially mitigated by the creation of a comprehensive human pangenome, previous work has shown that generating custom genomes is of value both to characterize the variation present and to serve as a more appropriate genomic reference. Here, we generated and characterized a custom complete genome assembly from KOLF2.1J. Mapping of sequencing reads to a personalized diploid assembly results in more comprehensive mapping compared to traditional linear references (i.e GRCh38). In addition, we provide a comprehensive custom gene annotation along with isoform expression and differential methylation analyses across multiple cell types. The assembly and all additional data is browsable and publicly available. This resource will enable more accurate investigation of the KOLF2.1J cell line and any genomics data generated compared to using traditional generalized references, while also serving as a foundational approach for establishing custom reference assemblies for other high-value iPSC lines.

genomics↗

bioRxiv2024

Assessing methylation detection for primary human tissue using Nanopore sequencing

DNA methylation most commonly occurs as 5-methylcytosine (5-mC) in the human genome and has been associated with human diseases. Recent developments in single-molecule sequencing technologies (Oxford Nanopore Technologies (ONT) and Pacific Biosciences) have enabled readouts of long, native DNA molecules, including cytosine methylation. ONT recently upgraded their Nanopore sequencing chemistry and kits from R9 to the R10 version, which yielded increased accuracy and sequencing throughput. However the effects on methylation detection have not yet been documented. Here we performed a series of computational analyses to characterize differences in Nanopore-based 5mC detection between the ONT R9 and R10 chemistries. We compared 5mC calls in R9 and R10 for three human genome datasets: a cell line, a frontal cortex brain sample, and a blood sample. We performed an in-depth analysis on CpG islands and homopolymer regions, and documented high concordance for methylation detection among sequencing technologies. The strongest correlation was observed between Nanopore R10 and Illumina bisulfite technologies for cell line-derived datasets. Subtle differences in methylation datasets between technologies can impact analysis tools such as differential methylation calling software. Our findings show that comparisons can be drawn between methylation data from different Nanopore chemistries using guided hypotheses. This work will facilitate comparison among Nanopore data cohorts derived using different chemistries from large scale sequencing efforts, such as the NIH CARD Long Read Initiative.

genomics↗