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Genomic Informatics in the Healthcare System
Published in Salvatore Volpe, Health Informatics, 2022
Shotgun sequencing is a significant improvement of DNA sequencing. In fact, the core concept of massive parallel sequencing used in NGS is adapted from shotgun sequencing. In general, the NGS sequencing process involves the preparation of a library of short DNA fragments through either enzymatic or sonication techniques. These short strands of DNA are then ligated to generic adapters in vitro. Polymerase chain reaction (PCR) amplification follows, performed using either emulsion PCR in oil-water emulsion micelles or bridge PCR on a solid surface coated with complementary primers. Subsequent sequencing of the amplicon (the portion of the DNA that has been replicated) is performed by either pyrosequencing, sequencing by ligation, or sequencing by synthesis. The large number of short reads generated from this process must then be aligned against a reference sequence. A plethora of software has been developed not only to align the reads but also to determine where deviations from a reference sequence exist. Many platforms, including the Illumina, Roche/454 FLX, the Solexa Genome Analyzer, and the Applied Biosystems SOLiD Analyzer, have been developed based on the aforementioned different methodologies in sequencing. These NGS platforms generate different base read lengths, error rates, and error profiles. NGS technologies have increased the speed and throughput capacities of DNA sequencing and, as a result, dramatically reduced overall sequencing costs and time.
Genetic Basis of Neuromuscular Disorders
Published in Maher Kurdi, Neuromuscular Pathology Made Easy, 2021
The single-gene approach typically involves the sequencing, using the chain termination method (CTM), of one gene at a time. Genomic DNA from the patient is prepared from blood, saliva, or buccal swabs. Then the gene of interest is amplified using polymerase chain reaction (PCR) and oligonucleotide primers targeting the regions of interest in the gene, which can be a region acting as a hotspot for mutations or all the coding exons including the splice-site junctions. The sequencing of the amplicons is performed using CTM on dedicated DNA analyzers. This approach is considered the gold standard of DNA sequencing due to the extremely low error rates in the process. However, the sequence output is limited, and it is informative only if a mutation is identified (Figure 8.1).
Tapping into the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Gold Mine for Individualization of Breast Cancer Treatment
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
Mark Abramovitz, Brian Leyland-Jones
FFPE tissue samples, a vast archive of pathologically well-characterized clinical samples from randomized trials, are an enormous potential resource that will allow translational scientists to more fully describe breast cancers at the molecular level and will ultimately have important etiologic and clinical implications. Even though the degradation of RNA that occurs because of the formalin fixation process results in an RNA species with an average size of approximately 200 nt (1), several groups have illustrated that it is feasible to extract and purify RNA from such fixed tissue and perform gene expression profiling (2–9). With the development of real-time quantitative (q) RT-PCR technology that has high detection sensitivity and high dynamic range, it is now possible to detect even rare messages in FFPE tissue and to examine the variation of expression over quite a large dynamic range. Amplicons are designed specifically on small segments of DNA [<100 base pairs (bp)] to achieve close to 100% efficiency for all amplicons, regardless of their length and nucleic acid composition. The potential of this technology becomes even more attractive in that it permits the analysis of thousands of tissue samples available through existing banks and without the need to collect the relatively complicated, freshly collected frozen tissue. Some studies have addressed improvements to the process of isolating high-quality FFPE RNA suitable for qRT-PCR or high-throughput gene expression profiling (10–13).
Baseline gut microbial profiles are associated with the efficacy of Bacillus subtilis and Enterococcus faecium in IBS-D
Published in Scandinavian Journal of Gastroenterology, 2023
Gaichao Hong, Ying Li, Min Yang, Gangping Li, Yu Jin, Hanhua Xiong, Wei Qian, Xiaohua Hou
DNA samples were quantified using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). DNA (30–50 ng) was used to generate amplicons using a MetaVx™ Library Preparation kit. V3, V4 and V5 hypervariable regions of prokaryotic 16S rDNA were selected for generating amplicons and taxonomy analysis. A panel of proprietary primers were designed aiming at relatively conserved regions bordering the V3, V4 and V5 hypervariable regions of 16S rDNA, which were amplified using forward primers (5′-CCTACGGRRBGCASCAGKVRVGAAT-3′) and reverse primers (5′-GGACTACNVGGGTWTCTAATCC-3′). First round PCR products were used as templates for second round amplicon enrichment PCR. At the same time, indexed adapters were added to the ends of the 16S rDNA amplicons to generate indexed libraries ready for downstream sequencing on Illumina Miseq. DNA libraries were validated by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), quantified by Qubit 2.0 Fluorometer, multiplexed and loaded into Illumina MiSeq to perform sequencing using a 2 × 300/250 paired-end (PE) configuration.
Targeted sequencing approach: Comprehensive analysis of DNA methylation and gene expression across blood and brain regions in suicide victims
Published in The World Journal of Biological Psychiatry, 2023
Katarina Kouter, Tomaž Zupanc, Alja Videtič Paska
Library was designed following an Illumina 16S protocol (modified accordingly) (Illumina 2013). Amplicons were generated using two rounds of PCR. During the first round, targeted regions were amplified (complete primer pair sequences in Supplementary Table S2). PCR reactions were carried out in a volume of 25 μL, using KAPA HiFi HotStart Uracil + ReadyMix (Roche, USA), 1 μM primers and 20 ng of DNA. PCR protocol was as followed: activation 5 min at 95 °C, primer pair dependent number of cycling (denaturation 30 s at 98 °C, annealing 15 s at primer pair dependent °C and extension 15 s at 72 °C), followed by final extension 1 min at 72 °C. Exact number of cycles and annealing temperatures can be found in Supplementary Table S3. Shorter unspecific amplifications were removed using AMPure XP beads (Beckman Coulter, USA). Amplicons of each subject were combined into an equimolar pool (concentrations were measured using Quant-iT PicoGreen dsDNA (Thermo Scientific, USA)).
Diagnostic performance of an in-house multiplex PCR assay and the retrospective surveillance of bacterial respiratory pathogens at a teaching hospital, Kelantan, Malaysia
Published in Pathogens and Global Health, 2023
Nik Mohd Noor Nik Zuraina, Suharni Mohamad, Habsah Hasan, Mohammed Dauda Goni, Siti Suraiya
Figure 1 shows the representative agarose gels with amplicons from clinical sputum specimens. The presence or absence of any target amplicons indicates positive or negative result by this assay, respectively. Of 200 clinical sputum specimens being tested, 79 specimens were found positive for single (n = 69) or multiple (n = 10) bacteria. The rest of 121 specimens was negative by this assay. Of these 79 specimens, total number of individual organisms detected by the multiplex PCR assay was 89, comprising of K. pneumoniae (n = 55), P. aeruginosa (n = 14), H. influenzae (n = 13), S. aureus (n = 6) and S. pneumoniae (n = 1). Results also found that all the 200 sputum specimens were negative for M. tuberculosis by this multiplex PCR assay. All the specimens of which positive for multiple bacteria consisted of K. pneumoniae in combination with other types of bacteria. Based on this multiplex PCR analysis, up to two types of the intended target bacteria were simultaneously detected from one single specimen. Detection of bacteria from clinical sputum specimens by PCR assay was compared with the results obtained from the gold standard sputum culture methods.