Long-read sequencing and optical mapping generates near T2T assemblies that resolves a centromeric translocation.

Long-read genome sequencing (lrGS) is a promising method in genetic diagnostics. Here we investigate the potential of lrGS to detect a disease-associated chromosomal translocation between 17p13 and the 19 centromere. We constructed two sets of phased and non-phased de novo assemblies; (i) based on lrGS only and (ii) hybrid assemblies combining lrGS with optical mapping using lrGS reads with a median coverage of 34X. Variant calling detected both structural variants (SVs) and small variants and the accuracy of the small variant calling was compared with those called with short-read genome sequencing (srGS). The de novo and hybrid assemblies had high quality and contiguity with N50 of 62.85 Mb, enabling a near telomere to telomere assembly with less than a 100 contigs per haplotype. Notably, we successfully identified the centromeric breakpoint of the translocation. A concordance of 92% was observed when comparing small variant calling between srGS and lrGS. In summary, our findings underscore the remarkable potential of lrGS as a comprehensive and accurate solution for the analysis of SVs and small variants. Thus, lrGS could replace a large battery of genetic tests that were used for the diagnosis of a single symptomatic translocation carrier, highlighting the potential of lrGS in the realm of digital karyotyping.

Discovery of non-reference processed pseudogenes in the Swedish population.

The vast majority of the human genome is non-coding. There is a diversity of non-coding features, some of which have functional importance. Although the non-coding regions constitute the majority of the genome, they remain understudied, and for a long time, these regions have been referred to as junk DNA. Pseudogenes are one of these features. A pseudogene is a non-functional copy of a protein-coding gene. Pseudogenes may arise through a variety of genetic mechanisms. Processed pseudogenes are formed through reverse transcription of mRNA by LINE elements, after which the cDNA is integrated into the genome. Processed pseudogenes are known to be variable across populations; however, the variability and distribution remains unknown. Herein, we apply a custom-designed processed pseudogene pipeline on the whole genome sequencing data of 3,500 individuals; 2,500 individuals from the thousand genomes dataset, as well as 1,000 Swedish individuals. Through these analyses, we discover over 3,000 pseudogenes missing from the GRCh38 reference. Utilising our pipeline, we position 74% of the detected processed pseudogenes-allowing for analyses of formation. Notably, we find that common structural variant callers, such as Delly, classify the processed pseudogenes as deletion events, which are later predicted to be truncating variants. By compiling lists of non-reference processed pseudogenes and their frequencies, we find a great variability of pseudogenes; indicating that non-reference processed pseudogenes may be useful for DNA testing and as population-specific markers. In summary, our findings highlight a great diversity of processed pseudogenes, that processed pseudogenes are actively formed in the human genome; and that our pipeline may be used to reduce false positive structural variation caused by the misalignment and subsequent misclassification of non-reference processed pseudogenes.