The Revised Diploid Genome Sequence of an Individual Human: An Optimized Assembly Workflow for Scaling of near Telomere-to-Telomere Assemblies

The first draft diploid genome assembly (HuRef) of an individual released in 2007 was a milestone in genomics. Here, we report HuRef2.0, a revision of HuRef assembled using a scalable two-step workflow employing only Oxford Nanopore Technologies (ONT) Simplex reads and the hifiasm assembler. Results are close in continuity to the recent telomere-to-telomere (T2T) assemblies, but were assembled from standard DNA samples without using multiple sequencing and mapping technologies, including ultra-long-reads and/or proximity-ligation. Three ONT flowcells ([~]103x coverage) from fresh blood DNA produced an assembly comprising 26 contigs, with gapless assembly of 23 chromosomes. Two gaps on chromosome (Chr)Y were locally assembled to yield the final T2T-assembly, HuRef2.0, with base accuracy >Q60 and 2,393 phase blocks with an NG50 value of 2.36 Mb. Assembly from a single ONT flowcell ([~]35x coverage) consistently produced an assembly more contiguous than GRCh38.p14, providing a foundation for further optimization and scaling. Assembly quality was assessed by direct chromosome-level alignments to reference genome, variant calling, and the annotation of gene-rich regions at Chr22q11, the extended MHC locus on Chr6, and several difficult to assemble regions of the genome, including the ribosomal RNA gene clusters, the sub-telomeric region on Chr4q35, and ChrY. More accurate but shorter Pacific Biosciences (PacBio) HiFi-reads produced less contiguous assemblies than from equivalent coverage of error-corrected ONT reads, indicating the importance of read-length. Finally, we compared HuRef2.0 to an assembly of an EBV-transformed lymphoblastoid cell line derived from the same donor. We observed no notable structural differences, indicating that low-passage archival transformed cells are reliable sources for genomic analysis.