The introduction of Next Generation Sequencing (NGS) technologies has enabled the establishment of significant new diagnostic applications in daily routine practice. Although the use of NGS technology in clinical diagnostics comes with challenges, its adoption is nevertheless inevitable due to the advantages it offers. In addition to extremely high sequencing capacity, NGS enables clonal sequencing of individual molecules, higher diagnostic sensitivity through parallel sequencing of entire gene panels, simplified handling, and parallel processing of samples, which is consequently more cost-effective. Recent technical advancements have made NGS a reliable alternative to classical Sanger sequencing.
Illumina sequencing-by-synthesis
The Illumina sequencing-by-synthesis (SBS) method was introduced in 2006 under the Solexa name. In this method, fragmented template DNA is covalently bound to a glass slide (FlowCell) via specific adapters, where the sequencing reaction takes place. Starting from the bound template molecule, clusters of identical molecules are formed through a PCR-like step called bridge amplification. Sequencing is performed cycle by cycle, utilizing reversible terminator chemistry and fluorescently labeled nucleotides. In each sequencing cycle, exactly one nucleotide complementary to the template DNA is incorporated. Subsequently, the fluorescent group is cleaved off, the emitted light signal is detected, and the terminator group is removed, allowing another nucleotide to be incorporated in the next cycle.
A unique feature of the Illumina SBS method is the ability to perform paired-end sequencing. In this approach, the DNA fragments to be sequenced are read from both ends, with a predetermined read length of 100–250 base pairs. Depending on the size of the DNA fragments, these reads may overlap or be separated by an unsequenced DNA portion (insert). Paired-end sequencing provides many advantages in bioinformatic analysis and can significantly improve the accuracy of analyses.
Several sequencing instruments use this method, including the Genome Analyzer IIx (GAIIx), its successor models in the HiSeq and NextSeq series, as well as benchtop sequencers such as MiSeq, MiniSeq, and iSeq. The throughput varies by device and is suitable for sequencing smaller gene panels as well as whole-exome and whole-genome approaches.
Illumina NovaSeq technology
Since February 2019, a dedicated NGS Core Facility has been available at the MVZ Martinsried. Equipped with an Illumina NovaSeq 6000 system, the latest generation of Illumina sequencing devices, it enables the production of genomic data at unprecedented scale and speed. With a flexible FlowCell setup, the NovaSeq system can efficiently perform multigene panel analyses, whole-exome analyses, and even whole-genome analyses.
The high throughput of the NovaSeq series is achieved through the use of so-called Patterned FlowCell technology. Unlike earlier MiSeq, NextSeq, or HiSeq devices, the DNA fragments to be sequenced do not bind randomly in clusters on the FlowCell. Instead, the surface of the FlowCell consists of billions of orderly nanowells, uniformly arranged at fixed distances from each other. A single DNA fragment can be sequenced in each of these nanowells. This structured organization offers several advantages over random cluster distribution:
●The organization of clusters in nanowells allows for more precise optimization of sequencing runs, reducing the risk of overloading the run.
●Signal detection and data analysis are faster by several hours, as signals can be assigned to specific nanowells.
●The risk of mixed signals caused by random clustering and insufficient spacing between clusters is eliminated, significantly increasing cluster density and sequencing throughput.
The NovaSeq 6000 uses the proven two-color sequencing-by-synthesis chemistry from the NextSeq line. As a result, run times of 25–44 hours can be achieved with throughput levels of up to 250–3000 gigabases per FlowCell. Initial read lengths are 2×150 bp paired-end reads, increasing to 2×250 bp through the use of the NovaSeq SB FlowCell. This enables the NovaSeq 6000 to address applications requiring extended read lengths, such as HLA typing, microbiome analyses, or HIV resistance testing.
By utilizing this technology, Medicover Genetics can maintain the highest standards of sequencing quality and quantity while further implementing its approach to integrated diagnostics.
Ion Torrent PGM
In early 2010, a new sequencing technology based on semiconductor technology was introduced by Ion Torrent Inc. This technique, now marketed and offered by Life Technologies, is also known as pH-mediated sequencing or “post-light” sequencing. The method follows a sequencing-by-synthesis approach, where a DNA template is complemented through sequential nucleotide incorporation. However, the detection method for the incorporated nucleotides substantially differs from previously described methods as it does not rely on optical signals. The incorporation of a nucleotide involves forming a covalent bond, releasing pyrophosphate and a positively charged hydrogen ion. In the Ion Torrent system, the nucleotide incorporation by DNA polymerase is detected by a change in pH caused by the released hydrogen ion. The DNA to be sequenced is placed in micro-reaction chambers on a semiconductor chip. These chambers contain DNA polymerase, and various nucleotides are added sequentially. Complementary nucleotides are incorporated by the polymerase, and the released hydrogen ions are detected by an ion-sensitive layer beneath the reaction chambers. Similar to the Roche 454 technology, multiple nucleotides may be incorporated into the template strand in cases of homopolymer regions. Here, too, the detected pH signal is proportional to the number of incorporated nucleotides.
Sequencing a semiconductor chip takes 2-4 hours, with read lengths depending on the kit used—averaging 100, 200, or 300 bp. Various chip sizes are available for flexible throughput: up to 10 Mb with the 314 chip, up to 100 Mb with the 316 chip, and up to 1 GB with the 318 chip.
Enrichment Methods
Since the data throughput generated by sequencing devices is enormous and not usually the limiting factor in most projects, genomic DNA samples are often prepared for sequencing without further preprocessing. However, in applications such as targeted resequencing of specific genes or gene panels related to the same disease indication, sequencing entire genomes is unnecessary. Consequently, various technologies for specific enrichment of target regions have been developed (target enrichment). Different PCR- or hybridization-based approaches are possible. Combining these methods with NGS can improve the analysis of disease-relevant gene sets in molecular genetic diagnostics by reducing time and costs. Utilizing the devices’ immense throughput allows multiple genes and samples to be analyzed together in a single run.
Applications
NGS methods are successfully applied in molecular genetic diagnostics. Clinical issues such as hereditary cardiomyopathy, connective tissue disorders, lung diseases, or neurological disorders can be efficiently and rapidly addressed through simultaneous sequencing of all genes relevant to the indication in a panel. Larger approaches, such as whole-exome sequencing, are useful in certain areas, such as intellectual disability, and have led to higher diagnostic rates in such cases.
Detecting minorities is particularly important in leukemia diagnostics, solid tumors, or complex pathogen spectra. In tumor diagnostics, mosaics, hard-to-access or limited materials such as biopsies, or treatment monitoring—where minimal residual disease needs to be detected—are common challenges. With sufficiently deep sequencing, NGS often allows identification of point mutations, deletions, amplifications, insertions, or gene rearrangements, even when the proportion of wild-type cells is relatively high. Sometimes allele discrimination is possible, enabling differentiation between different tumor clones. The potential applications extend far beyond the examples mentioned.
Molecular Karyotyping
The successful introduction of chromosomal microarray analyses for identifying submicroscopic structural aberrations in routine diagnostics, along with improvements in NGS-based methods (paired-end approach), will likely lead to the integration of molecular and cytogenetic diagnostics in the near future. High-resolution microarray platforms such as the Affymetrix CytoScan HD already allow for the detection of chromosomal imbalances at the level of a few kilobases. In the future, the use of NGS techniques will not only close the remaining gap to single-nucleotide resolution but also enable precise breakpoint determination of balanced chromosomal rearrangements.
Non-Invasive Prenatal Testing (NIPT)
The discovery of cell-free fetal DNA (cffDNA) in maternal blood opens the possibility of using NGS-based techniques for prenatal assessment of certain aneuploidies. Maternal and fetal DNA (over 10% fetal) are extracted from a blood sample from the expectant mother and sequenced. Quantification of the NGS sequences, combined with statistical analysis, can determine with high probability whether the fetus has trisomy of chromosome 21 (Down syndrome), 18 (Edwards syndrome), or 13 (Patau syndrome). This non-invasive testing can replace invasive techniques such as amniocentesis or chorionic villus sampling.
HLA Typing
Recent technological advancements have made NGS a reliable alternative to classical Sanger sequencing for HLA typing. NGS technology offers the advantage of clonal amplification of individual DNA molecules and simultaneous sequencing of various loci across a vast number of samples. Individual DNA molecules of a patient can be uniquely identified using molecular barcodes, known as index sequences, and assigned to specific samples. Higher sample throughput enables automation, significantly reducing labor time and costs.
Amplicon Strategy
Currently, NGS technology is used to type newly registered blood stem cell donors. Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1, and DPB1, as well as exon 6 and 7 of the ABO gene and exon 6 of the Rhesus factor, are sequenced using Illumina platforms (MiSeq, HiSeq, or NovaSeq). The amplicon strategy for enriching these gene segments combines “target generation” and “library preparation” in one process. The necessary PCR primers were developed in-house. To achieve high sample throughput, as many steps as possible have been automated. PCRs are pipetted in 384-well plates by a PCR machine (STARlet, Hamilton). Further steps like pooling, purification, and normalization are also automated on Hamilton devices. Raw data evaluation is conducted using Sequence Pilot software (JSI medical systems), which continuously matches sequences against the most important HLA database (IMGT/HLA Database). The resolution is high.
Long-Range Strategy
For donor-recipient pairing in upcoming stem cell transplants, the long-range strategy was introduced to achieve better resolution with fewer ambiguities. The HLA loci -A, -B, -C, -DRB1, -DQB1, and -DPB1 are fully amplified by PCR. Subsequent library preparation, where long PCR products are fragmented and indexed, is also automated on Hamilton devices. Data analysis is performed using GenDx’s NGSengine software, which is equipped with additional algorithms designed for this type of data.
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