Visualization and analysis of medically relevant tandem repeats in nanopore sequencing of control cohorts with pathSTR.

The lack of population-scale databases hampers research and diagnostics for medically relevant tandem repeats and repeat expansions. We attempt to fill this gap using our pathSTR web tool, which leverages long-read sequencing of large cohorts to determine repeat length and sequence composition in a healthy population. The current version includes 1040 individuals of the 1000 Genomes Project cohort sequenced on the Oxford Nanopore Technologies PromethION. A comprehensive set of medically relevant tandem repeats was genotyped using STRdust and LongTR to determine the tandem repeat length and sequence composition. PathSTR provides rich visualizations of this dataset and the feature to upload one's data for comparison along the control cohort. We demonstrate the implementation of this application using data from targeted nanopore sequencing of a patient with Myotonic Dystrophy type 1. This resource will empower the genetics community to get a more complete overview of normal variation in tandem repeat length and sequence composition and, as such, enable a better assessment of rare tandem repeat alleles observed in patients.

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.

A multiomic characterization of the leukemia cell line REH using short- and long-read sequencing.

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6::RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-yr-old at relapse, the cell line offers a practical model for the study of pediatric B-ALL. In recent years, short- and long-read DNA and RNA sequencing have emerged as a complement to karyotyping techniques in the resolution of structural variants in an oncological context. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole-genome sequencing, IsoSeq RNA sequencing, and short-read Illumina sequencing to create a detailed genomic and transcriptomic characterization of the REH cell line. Whole-genome sequencing clarified the molecular traits of disrupted ALL-associated genes including CDKN2A, PAX5, BTG1, VPREB1, and TBL1XR1, as well as the glucocorticoid receptor NR3C1 Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community.

MDM2 amplification in rod-shaped chromosomes provides clues to early stages of circularized gene amplification in liposarcoma.

Well-differentiated liposarcoma (WDLS) displays amplification of genes on chromosome 12 (Chr12) in supernumerary ring or giant marker chromosomes. These structures have been suggested to develop through chromothripsis, followed by circularization and breakage-fusion-bridge (BFB) cycles. To test this hypothesis, we compared WDLSs with Chr12 amplification in rod-shaped chromosomes with WDLSs with rings. Both types of amplicons share the same spectrum of structural variants (SVs), show higher SV frequencies in Chr12 than in co-amplified segments, have SVs that fuse the telomeric ends of co-amplified chromosomes, and lack interspersed deletions. Combined with the finding of cells with transient rod-shaped structures in tumors with ring chromosomes, this suggests a stepwise process starting with the gain of Chr12 material that, after remodeling which does not fit with classical chromothripsis, forms a dicentric structure with other chromosomes. Depending on if and when telomeres from other chromosomes are captured, circularized or linear gain of 12q sequences will predominate.

Mind the gap: the relevance of the genome reference to resolve rare and pathogenic inversions.

Both long-read genome sequencing (lrGS) and the recently published Telomere to Telomere (T2T) reference genome provide increased coverage and resolution across repetitive regions promising heightened structural variant detection and improved mapping. Inversions (INV), intrachromosomal segments which are rotated 180° and inserted back into the same chromosome, are a class of structural variants particularly challenging to detect due to their copy-number neutral state and association with repetitive regions. Inversions represent about 1/20 of all balanced structural chromosome aberrations and can lead to disease by gene disruption or altering regulatory regions of dosage sensitive genes in cis . Here we remapped the genome data from six individuals carrying unsolved cytogenetically detected inversions. An INV6 and INV10 were resolved using GRCh38 and T2T-CHM13. Finally, an INV9 required optical genome mapping, de novo assembly of lrGS data and T2T-CHM13. This inversion disrupted intron 25 of EHMT1, confirming a diagnosis of Kleefstra syndrome 1 (MIM#610253). These three inversions, only mappable in specific references, prompted us to investigate the presence and population frequencies of differential reference regions (DRRs) between T2T-CHM13, GRCh37, GRCh38, the chimpanzee and bonobo, and hundreds of megabases of DRRs were identified. Our results emphasize the significance of the chosen reference genome and the added benefits of lrGS and optical genome mapping in solving rearrangements in challenging regions of the genome. This is particularly important for inversions and may impact clinical diagnostics.