Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping.

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.

Enrichment of circulating trophoblasts from maternal blood using filtration-based Metacell® technology.

In a cell-based non-invasive prenatal test (cbNIPT), intact circulating trophoblasts (CTs) are isolated from maternal blood for subsequent genetic analysis. Enrichment of these CTs from maternal blood is the most challenging step in the cbNIPT workflow. This study aims to assess the suitability of the filtration-based Metacell® technology to enrich CTs from maternal blood at week 10 to 13 of gestation. The Metacell® technology is a novel size-based enrichment technology that combines blood filtration through 8 µm pores with an in vitro culture method. Three protocols were evaluated. First, 8 mL or 16 mL of maternal blood was filtered and subsequently cultured in vitro on the separation membrane for 3 days in RPMI 1640. In addition, 16 mL of maternal blood was filtered, and immediately processed without further culturing. Y-chromosome-specific qPCR or STR analysis was performed to evaluate the enrichment of CTs. A total of 44 samples from pregnant women, out of which 26 were carrying a male fetus, were processed. Although five enriched male fetus samples show detectable male DNA quantities, it cannot be excluded that the obtained positive signal is caused by cell-free fetal DNA sticking to the Metacell® separation membrane. In conclusion, the Metacell® technology, tested as described, is not suitable for consistent enrichment of CTs.

Nanopore sequencing of a forensic combined STR and SNP multiplex.

Nanopore sequencing for forensic purposes has gained attention, as it yields added discriminatory power compared to capillary electrophoresis (CE), without the need for a high up-front capital investment. Besides enabling the detection of iso-alleles, Massively Parallel Sequencing (MPS) facilitates the analysis of Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) in parallel. In this research, six single-contributor samples were amplified by such a combined multiplex of 58 STR and 94 SNP loci, followed by nanopore sequencing using an R10.3 flowcell. Basecalling was performed using two state-of-the-art basecallers, Guppy and Bonito. An advanced alignment-based analysis method was developed, which lowered the noise after alignment of the STR reads to a reference library. Although STR genotyping by nanopore sequencing is more challenging, correct genotyping was obtained for all autosomal and all but two non-autosomal STR loci. Moreover, genotyping of iso-alleles proved to be very accurate. SNP genotyping yielded an accuracy of 99% for both basecallers. The use of novel basecallers, in combination with the newly developed alignment-based analysis method, yields results with a pronouncedly higher STR genotyping accuracy compared to previous studies.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Digital Polymerase Chain Reaction for Assessment of Mutant Mitochondrial Carry-over after Nuclear Transfer for In Vitro Fertilization.

BACKGROUND: The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification. METHODS: We assessed the mutation load of 23 low-input human samples at the m.11778 locus, which is associated with Leber's hereditary optic neuropathy (LHON) using 2 droplet digital PCR platforms (Stilla Naica and Bio-Rad QX200) and the standard NGS strategy. Assay validation was performed by analyzing a titration series with mutation loads ranging from 50% to 0.01%. RESULTS: A good concordance in mutation rates was observed between both dPCR techniques and NGS. dPCR established a distinctly lower level of background noise compared to NGS. Minor alleles with mutation loads lower than 1% could still be detected, with standard deviations of the technical replicates varying between 0.07% and 0.44% mutation load. Although no significant systematic bias was observed when comparing dPCR and NGS, a minor proportional bias was detected. A slight overestimation of the minor allele was observed for the NGS data, most probably due to amplification and sequencing errors in the NGS workflow. CONCLUSION: dPCR has proven to be an accurate tool for the quantification of mitochondrial heteroplasmy, even for samples harboring a low mutation load (<1%). In addition, this alternative technique holds multiple benefits compared to NGS (e.g., less hands-on time, more straightforward data-analysis, and a lower up-front capital investment).

STRide probes: Single-labeled short tandem repeat identification probes.

The demand for forensic DNA profiling at the crime scene or at police stations is increasing. DNA profiling is currently performed in specialized laboratories by PCR amplification of Short Tandem Repeats (STR) followed by amplicon sizing using capillary electrophoresis. The need for bulky equipment to identify alleles after PCR presents a challenge for shifting to a decentralized workflow. We devised a novel hybridization-based STR-genotyping method, using Short Tandem Repeat Identification (STRide) probes, which could help tackle this issue. STRide probes are fluorescently labeled oligonucleotides that rely on the quenching properties of guanine on fluorescein derivatives. Mismatches between STRide probes and amplicons can be detected by melting curve analysis after asymmetric PCR. The functionality of the STRide probes was demonstrated by analyzing synthetic DNA samples for the D16S539 locus. Next, STRide probes were developed for five different CODIS core loci (D16S539, TH01, TPOX, FGA, and D7S820). These probes were validated by analyzing 13 human DNA samples. Successful genotyping was obtained using inputs as low as 31 pg of DNA, demonstrating high sensitivity. The STRide probes are ideally suited to be implemented in a microarray and present an important step towards a portable device for fast on-site forensic DNA fingerprinting.

Enrichment of circulating trophoblasts from maternal blood using laminar microscale vortices.

OBJECTIVE: Enrichment of circulating trophoblasts (CTs) from maternal blood at week 11-13 of gestation, using laminar microscale vortices, and evaluation of the performance of the VTX-1 Liquid Biopsy System in terms of CT recovery and purity. METHOD: Eight mililiter of blood was collected from 15 pregnant women and processed with the VTX-1 Liquid Biopsy System. Y-chromosome specific quantitative PCR was performed to estimate the number of enriched male CTs. To evaluate the VTX-1 performance, the target cell recovery was characterized by spiking experiments with a trophoblast cell line. Furthermore, the total quantity of DNA after enrichment was used to calculate the number of retained maternal cells. RESULTS: Successful recovery of male CTs was established in 7 out of 10 first trimester samples from pregnant women carrying a male fetus. The number of CTs, recovered from 8 ml of blood, was estimated between two and six. Spiking experiments resulted in a CT recovery of ±35 % with ±1524 retained maternal blood cells. CONCLUSION: CTs can be enriched from maternal blood with high purity, using laminar microscale vortices, starting from 8 ml of blood.

Kinship analysis on single cells after whole genome amplification.

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 pre-implantation 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 STR- and 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 high-quality 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.

Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling.

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.

Silicon µPCR Chip for Forensic STR Profiling with Hybeacon Probe Melting Curves.

The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-on-a-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 µl each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated on-chip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis.