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Expansions of short tandem repeats are genetic variants that have been20 implicated in neuropsychiatric and other disorders but their assessment 21 remains challenging with current molecular methods. Here, we developed a 22 Cas12a-based enrichment strategy for nanopore sequencing that, combined 23 with a new algorithm for raw signal analysis, enables us to efficiently target, 24 sequence and precisely quantify repeat numbers as well as their DNA 25 methylation status. Taking advantage of these single molecule nanopore 26 signals provides therefore unprecedented opportunities to study pathological 27 repeat expansions. 28 The expansion of unstable genomic Short Tandem Repeats (STRs) causes more 29 than 30 Mendelian human disorders 1 . For example, expansion of a GGGGCC-repeat 30 [(G 4 C 2 ) n ] within the C9orf72 gene is the most frequent monogenic cause of 31 Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS; 32 c9FTD/ALS; OMIM: # 105550) 2,3 . Similarly, accumulation of a CGG motif in the 33 FMR1 gene underlies the Fragile X Syndrome (FXS; OMIM # 300624), currently the 34 most common identifiable genetic cause of mental retardation and autism 4 . In both 35 prototypical repeat expansion disorders (Suppl. Discussion 1), recent evidence has 36 suggested pronounced inter-and intraindividual repeat variability as well as changes 37 in DNA methylation of the respective genomic regions to modulate disease 38 phenotype 5-8 . 39 To overcome current difficulties in characterizing expanded STRs (Suppl. Discussion 40 2) most notably we focused on three areas: i) optimization of Nanopore sequencing 41 and signal processing to capture STRs ii) development and implementation of a 42 3 target enrichment strategy to increase efficiency and iii) integration of expansion 43 measurements with DNA methylation of the same molecule. 44 45 Figure1 nanoSTRique: Generic repeat detection pipeline on raw nanopore signals. 46 a) Repeat quantification by signal-alignment of flanking prefix and suffix regions and HMM based 47 count on signal of interest. b) BioAnalyzer electropherogram, decoy alignment, RepeatHMM and 48 nanoSTRique counts of synthetic (G 4 C 2 ) n repeats (10k random reads per barcode, +/-10 % intervals 49 around expected repeat length). c) Nanopore sequencing and analysis of BAC clone 239 from a 50 c9ALS/FTD patient compared to cropped corresponding lane from Ref. 15 for illustration purpose. d) 51manual confirmation of detected repeat counts in synthetic repeats (n=16, 50, 49, 49, 47). 52 First, for benchmarking repeat expansion counting methods we constructed, verified 53 and nanopore sequenced plasmids with several synthetic (G 4 C 2 ) n -repeat lengths 9 . 54 We analyzed our results with currently available STR quantification pipelines 10,11 but 55 found those methods to become unreliable for more than 32 (G 4 C 2 ) n -repeats with 56 nanopore reads. To further improve the repeat analysis we developed a signal 57 processing algorithm for a more exact quantification of STR numbers in raw 58 na...

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For exact molecular diagnosis of CNS tumors, tumor tissue obtained through surgery is usually needed. However, knowledge on the molecular tumor type beforehand might influence surgical strategies and reduce surgery-associated risks. Molecular analyses of liquid biopsies have therefore gained increasing interest in order to secure diagnosis before surgery or to replace surgery in rare instances. Moreover, the minimally invasive nature of liquid biopsies enables a tight and longitudinal tumor monitoring with potentially higher sensitivity than imaging approaches. In this study, we applied Nanopore sequencing to 129 cell-free DNA (cfDNA) samples from the cerebrospinal fluid (CSF) of brain tumor patients, with 70.5% coming from pediatric patients. We analyzed copy number variations (CNV) and methylation patterns with a recently published random forest classifier (NanoDx). Circulating tumor DNA (ctDNA) was detected and successfully classified the tumor in 45% of all technically successful samples, both in pre- and early post-surgery samples as well as in samples from >14 days post-surgery, often with clinically unclear residual tumor or disease relapse. In all samples containing detectable tumor DNA, CNV analysis was more frequently successful than methylation analysis with overall detection rates of 88% and 44%, respectively. CNV analysis revealed diagnostic alterations, such as C19MC amplifications in ETMR as well as Chr.1q gain and Chr.6q loss in PFA ependymoma, which are important prognostic markers. Methylation analysis also allowed the classification of tumors with balanced genomes, like craniopharyngiomas. Finally, we were able to perform longitudinal analyses and found aberrations in the CNV profiles that were private to the tumor relapse, highlighting the potential of liquid biopsies to detect potentially relevant changes of tumor biology. Our results show that Nanopore sequencing is a promising approach to establish initial tumor diagnosis and to monitor disease courses by lumbar punctures.

Prader-Willi syndrome (PWS) is associated with severe hyperphagia, a specific behavioral phenotype and a high risk for developing psychotic episodes. Despite intense research, how genes within the PWS locus contribute to the phenotype remains elusive. In this study, we sequenced the whole genomes of 20 individuals with PWS using long-read nanopore sequencing by Oxford Nanopore Technologies (ONT). We demonstrate that ONT sequencing can resolve the PWS locus by determining the genetic subtype of PWS. Furthermore, we identified several novel structural variants (SV, >30bp) common in all PWS individuals. We are the first to show that the opioid system and the nociceptin/orphanin FQ system may be affected in PWS due to SVs in OPRM1 and OPRL1. Furthermore, we demonstrate that individuals with PWS, especially those with psychosis, exhibit a high burden of SVs in loci with known associations with bipolar disorder, schizophrenia and autism spectrum disorder. Our results challenge the current hypothesis that the PWS phenotype can be mainly explained by the loss of paternally expressed genes on chr15q11.2-13.

The intraoperative diagnosis of brain tumors remains a clinical challenge despite recent technological advances. The current clinical practice differentiates non-surgical brain tumors from those preferably treated with cytoreductive surgery employing intraoperative frozen section diagnostics. A detailed molecular diagnosis required for this classification task within the timeframe of a routine neurosurgical procedure is currently unavailable. We have analyzed a clinical cohort of several brain tumor entities using Nanopore long-read sequencing on two Oxford Nanopore Technologies sequencing platforms (MinION, PromethION). Since currently available molecular cancer classifiers such as the DKFZ methylation profiling classifier cannot be readily adapted to real-time sequencing analysis, we implemented a novel algorithm (MethyLYZR) to predict the underlying cancer type. Publicly available Illumina Infinium array data were used to train the classifier to distinguish 91 brain tumor classes. For validation of classification accuracy, we conducted a comprehensive validation strategy. Both nanopore platforms could sequence more than 5,000 pre-selected CpG within less than 20 minutes for most of our samples. When combining an optimized library preparation protocol with the time used for sequencing the minimal number of CpGs needed for classification, we saw sample-to-answer times of less than 1 hour – in many cases within 45 minutes - from receiving a fresh biopsy to a robust cancer type prediction. Comparing actual and predicted diagnoses resulted in a favorable error rate, indicating potentially highly clinical validity. Our real-time based molecular diagnostic algorithm enables, in most cases, a reliable diagnostic call within the timeframe of a typical neuro-oncological surgery. MethyLYZR as a predictive tool may allow us to adjust the surgical strategy and deliver the prognosis to our patients right after surgery, thus allowing for as-of-yet unexplored opportunities for the intraoperative application of individualized therapeutic modalities.

The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is functionally extinct, with only two females remaining alive. Efforts to rescue the NWR have inspired the exploration of unconventional conservation methods, including the generation of artificial gametes from induced pluripotent stem cells and somatic cell nuclear transfer. To enable the technologies required for these approaches, we used complementary sequencing and mapping methods to generate a NWR chromosome-level reference genome that meets or exceeds the metrics proposed by the Vertebrate Genome Project. It represents 40 autosomes, an X and a partially-resolved Y chromosome, and the mitochondrial genome. We compared the NWR reference genome to the southern white rhinoceros (SWR) population that has been physically separated from the NWR for tens of thousands of years. Using short-read data from the SWR and optical mapping, we found that the two populations are very similar on both the chromosome level and mitochondrial genome level. The results of this study are encouraging for the efforts underway to rescue the NWR.