Poster #7 - Krystal Kuang

Understanding the role of LINE-1 expression in Hematopoietic Stem Cell development using targeted long-read RNA sequencing

Krystal Kuang1, Dewan Shrestha2, Jinchuan Xing1,3, Yong Cheng2 1 Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ, USA 2 Department of Hematology, St. Jude's Children's Research Hospital, Memphis, TN, USA 3 Human Genetics Institute of New Jersey, Rutgers, The State University of New Jersey, Piscataway, NJ, USA

Long Interspersed Element-1 (LINE-1 or L1) is the only autonomous retrotransposon in the modern human genome, contributing to genetic diversity and genomic instability. However, studying L1 expression has been hampered by technical limitations. Specifically, short-read sequencing lacks the specificity to resolve evolutionarily young L1 loci due to their high sequence similarity, and conventional long-read RNA sequencing suffers from limited coverage because of the low level of L1 expression in most cells. To address these challenges, we developed a targeted long-read sequencing approach leveraging Oxford Nanopore Technology to enrich for young, active L1 elements. Using a panel of 54 custom-designed probes, we applied the protocol to human erythroid progenitor cells. We then developed a bioinformatics pipeline to detect young, active L1 transcripts. Reads were quality-checked and annotated with RepeatMasker, and low-confidence reads (<1kb in length, L1 >10% divergence from the consensus, etc.) were excluded to retain high-confidence reads containing L1 sequences. Resulting reads were mapped to a reference genome using Minimap2. To identify locus-specific expression and genomic context of active L1s, the high-confidence L1 loci were selected based on the sequence features (e.g., containing poly(A) tail), genomic contexts (e.g., no overlap with gene exons, reads start within L1 promoters, etc.). We further validated candidate L1 loci with Interactive Genome Viewer visualization. Preliminary results confirmed the expression of multiple high-confidence L1 loci, including those located within the genomic context of the hemoglobin subunit beta (HBB) gene. These results highlight the utility of targeted long-read approaches for precise characterization of retrotransposon activity and reveal transcription of young L1 elements in erythroid progenitors. Further long-read sequencing investigations may provide insight into how L1s influence HSC development and differentiation.