Forensic SNP genotyping using nanopore MinION sequencing

One of the latest developments in next generation sequencing is the Oxford Nanopore Technologies' (ONT) MinION nanopore sequencer. We studied the applicability of this system to perform forensic genotyping of the forensic female DNA standard 9947 A using the 52 SNP-plex assay developed by the SNPforID consortium. All but one of the loci were correctly genotyped. Several SNP loci were identified as problematic for correct and robust genotyping using nanopore sequencing. All these loci contained homopolymers in the sequence flanking the forensic SNP and most of them were already reported as problematic in studies using other sequencing technologies. When these problematic loci are avoided, correct forensic genotyping using nanopore sequencing is technically feasible

Performance of four modern whole genome amplification methods for copy number variant detection in single cells

Whole genome amplification (WGA) has become an invaluable tool to perform copy number variation (CNV) detection in single, or a limited number of cells. Unfortunately, current WGA methods introduce representation bias that limits the detection of small CNVs. New WGA methods have been introduced that might have the potential to reduce this bias. We compared the performance of PicoPLEX DNA-Seq (Picoseq), DOPlify, REPLI-g and Ampli-1 WGA for aneuploidy screening and copy number analysis using shallow whole genome massively parallel sequencing (MPS), starting from single or a limited number of cells. Although the four WGA methods perform differently, they are all suited for this application

STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods

The growing interest in liquid biopsies for cancer research and cell-based non-invasive prenatal testing (NIPT) invigorates the need for improved single cell analysis. In these applications, target cells are extremely rare and fragile in peripheral circulation, which makes the genetic analysis very challenging. To overcome these challenges, cell stabilization and unbiased whole genome amplification are required. This study investigates the performance of four WGA methods on single or a limited number of cells after 24 hour of Streck Cell-Free DNA BCT preservation. The suitability of the DNA, amplified with Ampli1, DOPlify, PicoPLEX and REPLI-g, was assessed for both short tandem repeat (STR) profiling and copy number variant (CNV) analysis after shallow whole genome massively parallel sequencing (MPS). Results demonstrate that Ampli1, DOPlify and PicoPLEX perform well for both applications, with some differences between the methods. Samples amplified with REPLI-g did not result in suitable STR or CNV profiles, indicating that this WGA method is not able to generate high quality DNA after Streck Cell-Free DNA BCT stabilization of the cells

Performance of a TthPrimPol-based whole genome amplification kit for copy number alteration detection using massively parallel sequencing

Starting from only a few cells, current whole genome amplification (WGA) methods provide enough DNA to perform massively parallel sequencing (MPS). Unfortunately, all current WGA methods introduce representation bias which limits detection of copy number aberrations (CNAs) smaller than 3 Mb. A recent WGA method, called TruePrime single cell WGA, uses a recently discovered DNA primase, TthPrimPol, instead of artificial primers to initiate DNA amplification. This method could lead to a lower representation bias, and consequently to a better detection of CNAs. The enzyme requires no complementarity and thus should generate random primers, equally distributed across the genome. The performance of TruePrime WGA was assessed for aneuploidy screening and CNA analysis after MPS, starting from 1, 3 or 5 cells. Although the method looks promising, the single cell TruePrime WGA kit v1 is not suited for high resolution CNA detection after MPS because too much representation bias is introduced

Short tandem repeat analysis after whole genome amplification of single B-lymphoblastoid cells

To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Unfortunately, studies comparing different WGA methods for downstream human identification Short Tandem Repeat (STR) analysis remain absent. Therefore, the aim of this work was to assess the performance of four commercially available WGA kits for downstream human identification STR profiling on a B-lymphoblastoid cell line. The performance was assessed using an input of one or three micromanipulated cells. REPLI-g showed a very low dropout rate, as it was the only WGA method in this study that could provide a complete STR profile in some of its samples. Although Ampli1, DOPlify and PicoPLEX did not detect all selected STR markers, they seem suitable for genetic identification in single-cell applications

Massively parallel sequencing of micro-manipulated cells targeting a comprehensive panel of disease-causing genes : a comparative evaluation of upstream whole-genome amplification methods

Single Gene Disorders (SGD) are still routinely diagnosed using PCR-based assays that need to be developed and validated for each individual disease-specific gene fragment. The TruSight One sequencing panel currently covers 12 Mb of genomic content, including 4813 genes associated with a clinical phenotype. When only a limited number of cells are available, whole genome amplification (WGA) is required prior to DNA target capture techniques such as the TruSight One panel. In this study, we compared 4 different WGA methods in combination with the TruSight One sequencing panel to perform single nucleotide polymorphism (SNP) genotyping starting from 3 micro-manipulated cells. This setting simulates clinical settings such as day-5 blastocyst biopsy for Preimplantation Genetic Testing (PGT), liquid biopsy of circulating tumor cells (CTCs) and cancer-cell profiling. Bulk cell samples were processed alongside these WGA samples to serve as a performance reference. Target coverage, coverage uniformity and SNP calling accuracy obtained using any of the WGA, is inferior to the results obtained on bulk cell samples. However, results after REPLI-g come close. Compared to the other WGA methods, the method using REPLI-g WGA results in a better coverage of the targeted genomic regions with a more uniform read depth. Consequently, this method also results in a more accurate SNP calling and could be considered for clinical genotyping of a limited number of cells

Hyperbolic Metamaterial Nanoparticles for Efficient Hyperthermia in the II and III Near-Infrared Windows

The use of gold nanoparticles for hyperthermia therapy in near infrared (NIR) spectral regions has catalysed substantial research efforts due to the potential impact in clinical therapy applications. However, the photoscattering effect scaling with the square of the nanoparticle volume leads to a low absorption efficiency, which has hindered the utility of gold nanoparticles in NIR II regions above 1000 nm. Here, we conquer this limit by introducing hyperbolic metamaterial nanoparticles that are made of multi-layered gold/dielectric nanodisks and exhibit >70% absorption efficiency in the NIR II and III regions. Their high light-to-heat conversion is demonstrated by a much larger temperature increase than that of gold nanodisks with the same amount of gold. Efficient in vitro hyperthermia of living cells with negligible cytotoxicity shows the potential of our approach for next-generation bio-medical applications

Whole genome amplification with SurePlex results in better copy number alteration detection using sequencing data compared to the MALBAC method

Current whole genome amplification (WGA) methods lead to amplification bias resulting in over-and under-represented regions in the genome. Nevertheless, certain WGA methods, such as SurePlex and subsequent arrayCGH analysis, make it possible to detect copy number alterations (CNAs) at a 10 Mb resolution. A more uniform WGA combined with massive parallel sequencing (MPS), however, could allow detection at higher resolution and lower cost. Recently, MALBAC, a new WGA method, claims unparalleled performance. Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation. Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples. An even more uniform coverage was observed in samples following a PCR-free library preparation. In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material

On-Demand Intracellular Delivery of Single Particles in Single Cells by 3D Hollow Nanoelectrodes

Delivery of molecules into intracellular compartments is one of the fundamental requirements in molecular biology. However, the possibility of delivering a precise number of nano-objects with single-particle resolution is still an open challenge. Here we present an electrophoretic platform based on 3D hollow nanoelectrodes to enable delivery of single nanoparticles into single selected cells and monitoring of the single-particle delivery by surface-enhanced Raman scattering (SERS). The gold-coated hollow nanoelectrode capable of confinement and enhancement of electromagnetic fields upon laser illumination can distinguish the SERS signals of a single nanoparticle flowing through the nanoelectrode. Tight wrapping of cell membranes around the nanoelectrodes allows effective membrane electroporation such that single gold nanorods are delivered on demand into a living cell by electrophoresis. The capability of the 3D hollow nanoelectrodes to porate cells and reveal single emitters from the background in continuous flow is promising for the analysis of both intracellular delivery and sampling