Human genome sequencing and assembly from Pacific Biosciences HiFi reads
We continue to develop and improve scalable workflows for telomere-to-telomere (T2T) genome assemblies. Here, we report HuRef2, a revision of HuRef, using Oxford Nanopore Technologies (ONT) simplex reads and the hifasm assembler. The results are close in continuity to the recent T2T assemblies, but were assembled from typical DNA from fresh or archival tissues without the concomitant use of multiple sequencing and mapping technologies, including the need for ultra-long reads. The workflow is hi-throughput, low-cost, and requires little or no post-assembly polishing, thus providing a foundation for scaling. Assembly from 100x ONT reads from fresh blood, comprised 26 contigs with a contig N50 value of 154.7 Mb, >Q66 base accuracy, and the complete gapless assemblies of 23 chromosomes. Two gaps on chromosome-Y were filled by manual curation to a T2T assembly. Similar coverage in Pacific Biosciences (PacBio) HiFi reads yield a less contiguous assembly (JCVblood-PB) comprising 67 contigs with gapless assemblies of 17 chromosomes. Assembly qualities were assessed by chromosome-to-chromosome alignments to reference genomes, annotation of a 11 Mb gene rich region of chromosome 6 that includes the extended HLA locus, and at several difficult to assemble regions of the genome, including the ribosomal RNA gene clusters, the macro-satellite sub-telomeric region on chromosome 4q35, and the Y-chromosome. We also compared the assembled genomes from blood to that of an EBV-transformed lymphoblast cell-line derived from the same donor, where we observed no notable structural differences, indicating low-passage archival transformed cells could be a reliable source for genomic structural analysis.