Nanopore sequencing for forensic short tandem repeats (STR) genotyping comes with the advantages associated with massively parallel sequencing (MPS) without the need for a high up-front device cost, but genotyping is inaccurate, partially due to the occurrence of homopolymers in STR loci. The goal of this study was to apply the latest progress in nanopore sequencing by Oxford Nanopore Technologies in the field of STR genotyping. The experiments were performed using the state of the art R9.4 flow cell and the most recent R10 flow cell, which was specifically designed to improve consensus accuracy of homopolymers. Two single-contributor samples and one mixture sample were genotyped using Illumina sequencing, Nanopore R9.4 sequencing, and Nanopore R10 sequencing. The accuracy of genotyping was comparable for both types of flow cells, although the R10 flow cell provided improved data quality for loci characterized by the presence of homopolymers. We identify locus-dependent characteristics hindering accurate STR genotyping, providing insights for the design of a panel of STR loci suited for nanopore sequencing. Repeat number, the number of different reference alleles for the locus, repeat pattern complexity, flanking region complexity, and the presence of homopolymers are identified as unfavorable locus characteristics. For single-contributor samples and for a limited set of the commonly used STR loci, nanopore sequencing could be applied. However, the technology is not mature enough yet for implementation in routine forensic workflows.

CYP2D6 is a very important pharmacogene as it is responsible for the metabolization or bioactivation of 20 to 30% of the clinically used drugs. However, despite its relatively small length of only 4.4 kb, it is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants.

Short Tandem Repeat (STR-) and Single Nucleotide Polymorphism (SNP-) genotyping have been extensively studied within forensic kinship analysis. Nevertheless, no results have been reported on kinship analysis after whole genome amplification (WGA) of single cells. This WGA step is a necessary procedure in several applications, such as cell-based non-invasive prenatal testing (cbNIPT) and preimplantation genetic diagnosis (PGD). In cbNIPT, all putative fetal cells must be discriminated from maternal cells after enrichment from whole blood. This study investigates the efficacy and evidential value of STRand SNP-genotyping methods for the discrimination of 24 single cells after WGA, within three families. Formaldehyde-fixed and unfixed cells are assessed in offspring-parent duos and offspring-mother-father trios. Results demonstrate that both genotyping methods can be used in all tested conditions and scenarios with 100% sensitivity and 100% specificity, with a similar evidential value for fixed and unfixed cells. Moreover, sequence-based SNP-genotyping results in a higher evidential value than length-based STR-genotyping after WGA, which is not observed using highquality offspring bulk DNA samples. Finally, it is also demonstrated that the availability of the DNA genotypes of both parents strongly increases the evidential value of the results. For many years, length-based Short Tandem Repeat (STR-) genotyping has been the golden standard in paternity testing and forensic kinship analysis 1,2. However, the benefit of sequence-based Single Nucleotide Polymorphism (SNP-) genotyping combined with STR-genotyping has recently been established in complex kinship cases 3-5. For example, Mendelian incompatibilities in biologically true paternity cases are less likely to occur using SNPmarkers, due to their lower mutation rate compared to STRs, 10-8 versus 10-3 , respectively 6. This lower mutation rate results in a more stable inheritance of SNPs over generations, which is especially valuable in parentage cases. Furthermore, SNP-markers are more suitable than STR-markers for the analysis of fragmented DNA samples, as the SNP-amplicons are smaller compared to the lengthy STR-amplicons 7. Despite the theoretical advantages of SNPs over STRs in forensic casework, there has been much debate whether SNPs will ever fully replace STRs 8. A considerable disadvantage of SNPs is their less polymorphic nature. Since most SNPs are bi-allelic in the population, 40 or more SNPs are required to provide a similar discriminative power as 13-15 STRs for identification purposes 9-11. For kinship analysis, even more SNPs are required as shared alleles rather than shared genotypes are examined amongst relatives 11. Phillips et al. stated that on average 60 forensically relevant SNPs are required to match the power of STRs for kinship analysis 11 , while Ayres et al. demonstrated that 50-60 SNPs are required in offspring-mother-father trios, and 70-80 SNPs are required in offspring-parent duos 10. Due to the emerging interest in SNP-genotyping wi...

BackgroundSystemic sclerosis (SSc) and primary biliary cholangitis (PBC) are autoimmune diseases that may occur concomitantly and are both strongly associated with disease-specific autoantibodies. This study investigated the prevalence and fine specificity of PBC-specific serology (PBC-Ab) and associations with the SSc-subtypes and SSc-specific antibodies as well as the association with cholestatic liver enzymes. Furthermore, three different techniques for the detection of PBC-Ab were compared.MethodsSerum of 184 Belgian SSc patients with a known SSc-antibody profile, was analyzed for PBC-Ab (antimitochondrial antibodies [AMA], anti-Gp210, anti-Sp100 and anti-PML) using indirect immunofluorescence (IIF) analysis on human epithelioma-2000 (HEp-2000) cells (ANA-IIF, Immunoconcepts) and liver-kidney-stomach tissue sections (IIF-LKS) (Menarini), and a line immunoblot (LB) (EuroImmun). Alkaline phosphatase/γ-glutamyl transferase (ALP/GGT) were evaluated at time of first sampling (t0) and after 3 years of follow-up (t3).ResultsPBC-Ab were present in 13% of patients and significantly correlated with centromere antibodies (anti-CENP-B), but not correlated with the limited cutaneous SSc subgroup (lcSSc). The most frequent reactivities were AMA (11%, with 9% AMA-M2) and Sp-100 antibodies (5%), showing a major overlap. There was no relevant association between the presence of PBC-Ab and ALP or GGT elevation at t0 nor at t3. Detection of AMA with IIF-LKS is comparable to LB. ANA-IIF screening was less sensitive compared to LB.ConclusionsA wide range of PBC-Ab is detectable in SSc in the absence of cholestatic liver enzyme elevations, even after 3 years of follow-up. However, as these antibodies may precede PBC-disease up to 10 years further prospective follow-up of our cohort will be necessary.

The currently used pharmacogenetic genotyping assays offer limited haplotype information, which can potentially cause specific functional effects to be missed. This study tested if Targeted Locus Amplification (TLA), when using non-patient-specific primers combined with Illumina or Nanopore sequencing, can offer an advantage in terms of accurate phasing. The TLA method selectively amplifies and sequences entire genes based on crosslinking DNA in close physical proximity. This way, DNA fragments that were initially further apart in the genome are ligated into one molecule, making it possible to sequence distant variants within one short read. In this study, four pharmacogenes, CYP2D6, CYP2C19, CYP1A2 and BRCA1, were sequenced after enrichment using different primer pairs. Only 24% or 38% of the nucleotides mapped on target when using Illumina or Nanopore sequencing, respectively. With an average depth of more than 1000X for the regions of interest, none of the genes were entirely covered with either sequencing method. For three of the four genes, less than half of the variants were phased correctly compared to the reference. The Nanopore dataset with the optimized primer pair for CYP2D6 resulted in the correct haplotype, showing that this method can be used for reliable genotyping and phasing of pharmacogenes but does require patient-specific primer design and optimization to be effective.