Precise identification of Higher Order Repeats (HORs) in T2T-CHM13 assembly of human chromosome 21 – novel 52mer HOR and failures of hg38 assembly

Abstract From the complete T2T-CHM13 assembly of human chromosome 21, recently sequenced by the T2T Consortium, the precise alpha satellite higher-order repeat (HOR) structure is computed using novel high-precision GRM2023 algorithm, which includes Global Repeat Map (GRM) and Monomer Distance (MD) diagrams. This study rigorously identified and structurally analyzed alpha satellite HORs within the centromere. The major alpha satellite HOR array in chromosome 21 reveals a novel Cascading 11mer HOR copies with subfragments of periods 7, 4 and 20, 9. Within each row in the cascading HOR, the monomers are of different types, but different rows within the same cascading 11mer HOR can contain more than one monomer of the same type. Each canonical 11mer HOR copy comprises 11 monomers yet belong to 10 different monomer types. Another pronounced 23/25mer HOR array is of mixed Willard's/Cascade HOR type. It was found that the 33mer HOR and major 8mer HOR identified previously in hg38 assembly are absent in the T2T-CHM13 assembly, pointing inadequacies of hg38 assembly. The novel 52mer HOR was discovered, with the longest alpha satellite HOR copy in human genome. Previous results for alphoid subfamilies identified using restriction enzymes mostly align with precise predictions for HORs and/or subfragments obtained by applying the GRM2023 algorithm to the complete T2T-CHM13 assembly. Alphoid subfamilies previously identified by restriction enzymes approximately correspond either to a nmer HOR or to a subfragment align with the framework of Cascading HORs.