Preimplantation Genetic Testing for Aneuploidies: Where We Are and Where We're Going
Darren K. Griffin, Gary L. Harton in Preimplantation Genetic Testing, 2020
Sequencing is the process of determining the order of nucleotides. The “next-generation” designation refers to the more recently developed technologies that have the ability to generate this information quickly, accurately, and in a cost-effective manner. These methods started emerging in the early 2000s and catapulted the fields of biological research and medical genetics into a new era. They offer a major progression toward a more comprehensive characterization of the human genome and associated genetic disease. The field of PGT-A began harnessing these tools for routine use a decade later [76–78]. The most popular sequencing platforms utilized for PGT-A purposes include Illumina and Ion Torrent technologies. Both systems essentially entail single-stranded DNA template being repeatedly exposed to a sequence of dNTPs. Incorporation of bases complementary to the template can be detected in different ways. Illumina sequencing by synthesis technology enriches DNA templates with fluorescently labeled chain-terminating nucleotides, and base incorporation/calling is determined by light detection using specialized cameras. Ion Torrent/LifeTech technology, rather than utilizing optical components, detects nucleotide sequences by changes in pH, as nucleotide incorporation releases protons, changing the pH of the surrounding solution proportional to the number of incorporated nucleotides.
Genomic Informatics in the Healthcare System
Salvatore Volpe in Health Informatics, 2022
DNA is the code of all biological life on earth. Humans have sought to unravel its mysteries so that the origins of life itself may be revealed. The first sequencing methodology, known as Sanger sequencing, uses specifically manipulated nucleotides to read through a DNA template during DNA synthesis. This sequencing technology requires a specific primer to start the read at a specific location along the DNA template and record the different labels for each nucleotide within the sequence up to 1000–1200 base pairs (bps). Subsequently, an approach called shotgun sequencing was developed for longer read of sequences. In this approach, genomic DNA is enzymatically or mechanically broken down into smaller fragments and cloned into sequencing vectors in which cloned DNA fragments can be sequenced individually. The complete sequence of a long DNA fragment can be eventually generated by these methods by alignment and reassembly of sequence fragments based on partial sequence overlaps.
Preimplantation Genetic Testing
Steven R. Bayer, Michael M. Alper, Alan S. Penzias in The Boston IVF Handbook of Infertility, 2017
Next-generation sequencing (NGS) is a technology that uses optimized, high-throughput DNA amplification to sequence DNA. The process involves fragmenting DNA into millions of small fragments that are then fused with an adaptor and a barcode to create a DNA library. The library is then loaded into a flow cell where the fragments bind to a surface of complementary surface-bound oligonucleotides and then amplified to create distinct clonal clusters. High-throughput, paired-end reversible terminator-based sequencing of the fragments detects single bases as they are incorporated into the DNA template strands, reducing sequencing errors. Paired-end sequencing produces twice the number of reads that occur and the paired sequences are aligned as read pairs, further reducing the likelihood of errors in sequencing. The amplified fragments are aligned to a reference genome to detect differences between the fragment and the reference. The attachment of the barcode allows for multiple libraries to be run simultaneously and then sorted before final analysis. Advances in NGS have reduced time for library preparation and time for sequencing. The ability to multiplex allows for scalable instrumentation depending on the anticipated utilization. NGS will detect whole chromosome aneuploidy, mosaicism, triploidy, large deletions, or duplications greater than 50 Mb, some clinically significant deletions or duplications 800 b to 1 Mb, uniparental disomy, and mitochondrial copy number.
Cytogenetic and molecular genetic methods for chromosomal translocations detection with reference to the KMT2A/MLL gene
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Nikolai Lomov, Elena Zerkalenkova, Svetlana Lebedeva, Vladimir Viushkov, Mikhail A. Rubtsov
Illumina and Ion Torrent compete in the field of short-read sequencing with the sequencing platform produced by MGI (Shenzhen, China) that utilizes the technology developed by Complete Genomics (San Jose, California). During template preparation, the fragments are amplified using the rolling circle amplification, instead of PCR. The resulting concatemers form DNA nanoballs (DNBs), which are placed into the flow cell. Each DNB rests in a separate section of the patterned flow cell [90]. The MGI instrument performs sequencing using the combinatorial probe-anchor synthesis (cPAS) technology. The sequencing process consists of three steps: the addition of a labeled terminated nucleotide, the detection of the added nucleotide by fluorescently labeled monoclonal antibodies, and the cleavage of a terminator [91]. The sequencing reads produced by the MGI platform can reach up to 400 nt in length (or 150 nt in the paired-end sequencing). However, 400 nt reads come at the cost of sequencing time; for example, sequencing the entire human genome can take several days. The MGI platform covers the range of applications that other short-read sequencing platforms have, but with a lower sequencing cost [92–97].
Genetic screening as an adjunct to universal newborn hearing screening: literature review and implications for non-congenital pre-lingual hearing loss
Published in International Journal of Audiology, 2019
Christine D’Aguillo, Sara Bressler, Denise Yan, Rahul Mittal, Robert Fifer, Susan H. Blanton, Xuezhong Liu
Direct sequencing is used to determine the exact order of nucleotide bases in a given gene or region of interest, typically 1000 base pairs in length (Linden Phillips et al. 2013). The most widely used method is Sanger Sequencing. The advantage of this method is that it is able to identify almost all mutations present in a sequence, including novel mutations, and is considered the most accurate. However, this method is the most time consuming, labour intensive, and expensive. Thus, this method is now typically used to identify novel mutations or to verify results from an experimental screening technology. This review identified several papers that used direct sequencing as the primary screening mechanism, but these studies generally screened large populations for 1–5 mutations in only 1–3 genes (Zaputovic et al. 2008; Schimmenti et al. 2011; Wang et al. 2011; Iwata et al. 2013; Minami et al. 2013; Kim et al. 2013; Dai et al. 2015).
Management of antithrombin deficiency: an update for clinicians
Published in Expert Review of Hematology, 2019
Carlos Bravo-Pérez, Vicente Vicente, Javier Corral
Based on the percentage of positive findings and the cost, we proposed an algorithm for genetic analysis of cases with antithrombin deficiency (Figure 1) [13]. The first step consists of sequencing by Sanger method the seven exons and flanking regions of SERPINC1, what identifies molecular alterations in the majority of subjects (up to 70–80%). Massive Next Generation Sequencing (NGS) methods may be alternatively used and will replace Sanger sequencing soon. After that, for cases with negative results, Multiple Ligand Probe Amplification (MLPA) could be used to detect gross gene defects, which constitutes 2–5% of cases [62,63]. We have developed a simple method that detects the relatively common tandem duplication of exon 6, which is hardly detected by other molecular methods [64]. Whole SERPINC1 sequencing might reveal mutations in non-coding regions, such as the promoter or regulatory sequences, which also represent a small proportion of cases [65,66]. Finally, cases with negative findings in SERPINC1 may be tested for glycosylation defects, as this mechanism may underline up to 27% of these cases [39]. Following this algorithm, in the largest series of patients, including ours, a molecular basis is identified in near 85% of cases [13].
Related Knowledge Centers
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