Genomic Informatics in the Healthcare System
Salvatore Volpe in 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.
Beneficial Lactic Acid Bacteria
K. Balamurugan, U. Prithika in Pocket Guide to Bacterial Infections, 2019
Some methods of molecular typing are based on use of oligonucleotide primers complementary to repetitive sequences. Diverse regions of DNA flanked by the rep sequences are amplified, leading to amplicon patterns. The conserved repetitive sequences are divided into four types: the repetitive extragenic palindromic (REP), the enterobacterial repetitive intergenic consensus (ERIC), the BOX, and the polytrinucleotide (GTG)5 sequences (Mohapatra et al. 2007). Repetitive extragenic palindromic (REP) primers, the enterobacterial repetitive intergenic consensus (ERIC) primers, and the (GTG)5 primer can be used in the typing of Lactobacillus strains. DNA concentration and quality did not affect the ERIC-PCR profiles, indicating that this method, unlike other high-resolution methods, can be adapted to high-throughput analysis of isolates. Also, ERIC-PCR simultaneously types isolates to the strain and species levels, compared to PFGE that can type only to the strain level (Stephenson et al. 2009). PCR using the BOXAIR primers provides differentiation at species, subspecies and strain level, acting as the tool confirming phenotypic identification (Mohammed et al. 2009).
Genetic analysis of the embryo
David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham in Textbook of Assisted Reproductive Techniques, 2017
Single-cell molecular analysis for PGD was made possi- ble by PCR, which was first introduced in the mid-1980s. The technique enriches a DNA sample for one specific oligonucleotide fragment, the PCR product or amplicon. The technique uses a pair of short oligonucleotide frag- ments—primers—that are homologous to stretches of genomic DNA at a locus of interest. The PCR thermocy- cler is programmed to perform successive cycles consist- ing of denaturation at temperatures >90°C, during which the double-stranded template DNA melts into two sepa- rate single strands; annealing, in which the primers attach to their region of homology; and extension, during which new nucleotides (dNTPs) are added in succession to rec- reate a double-stranded DNA molecule by the enzymatic action of the thermostable Taq polymerase. The resulting new strands serve as templates for the subsequent cycles. After 30-40 such cycles, the initial minute quantity of DNA is amplified to the extent that it can actually be visu- alized by methods such as radioactive labeling, ethidium bromide, or silver staining. The PCR products may be further subjected to a variety of analytic techniques that determine the presence of point mutations, small deletions or insertions, or for analysis of linked polymorphic genetic markers. Finally, the precise composition of the amplified fragment may be studied by direct sequencing.
Aberrant enteric neuromuscular system and dysbiosis in amyotrophic lateral sclerosis
Published in Gut Microbes, 2021
Yongguo Zhang, Destiny Ogbu, Shari Garrett, Yinglin Xia, Jun Sun
Genomic DNAs were PCR-amplified with the Earth Microbiome Project primers CS1_515 F and CS2_806 R targeting the V3–V4 regions of the 16S rRNA gene. A two-stage “targeted amplicon sequencing” protocol as described in31 was used to generate amplicons. Two independent PCR steps were conducted in the workflow for preparing samples for next-generation amplicon sequencing. In the first stage, PCR amplification was performed using primers containing CS1 and CS2 linkers (CS1_341 F: 50-ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGTAA-30; CS2_806 R: 50-TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTCTAAT-30) to the V3–V4 variable region of the 16S rRNA gene, while in the second stage, PCR amplification was performed on the first stage of PCR products using the Fluidigm Access Array barcoded primers. The 16S rRNA gene sequencing was performed using MiSeq according to the Illumina protocol. Based on the distribution of reads per barcode, the amplicons were pooled, re-pooled and purified. Then, the re-pooled libraries were loaded onto a Miniseq flow cell and sequenced (2x153 paired-end reads). Library preparation, pooling, and sequencing were performed at the Genome Research Core within the Research Resources Center at the University of Illinois Chicago.
Point-by-Point Progress: Gonorrhea Point of Care Tests
Published in Expert Review of Molecular Diagnostics, 2020
Charlotte A. Gaydos, Johan H. Melendez
The first commercial assay to employ NAAT technology and receive FDA clearance is the GeneXpert® assay (Cepheid, Sunnyvale, CA). This assay offers near-patient tests, combining microfluidic technology with real-time PCR. The cartridge-based assay extracts and amplifies the DNA, and detects the PCR amplicon in 90 minutes. Sensitivities and specificities ranged from 95.6 to 100% and 99.9 to 100%, respectively for both female (vaginal, cervical, and urine) and male (urine) specimens [32]. Additionally, research indicates there were no false-positive results with other Neisseria spp. or other genital near neighbors [33]. The WHO has also confirmed the sensitivity and specificity of this assay with a global selection of Ng isolates, finding high performance [34]. Although the assay requires 90 minutes which is a bit longer than ideal for some situations, it has been successfully used at some clinical encounters, especially when samples could be obtained before seeing the clinician. This has been shown in the widely-cited Dean Street Express (DSE) Clinic study [35]. The DSE Clinic in London has used the rapid Xpert® assay extensively and has demonstrated that the rapid testing express visits resulted in a reduced mean time to notification of results from 8.68 days at the Dean Street clinic compared to 0.27 days for the DSE.
Critical insight into recombinase polymerase amplification technology
Published in Expert Review of Molecular Diagnostics, 2022
Kary Mullis developed the revolutionary technique of polymerase chain reaction (PCR) in 1983 [1], and its modern version has evolved through several of the improvements in the chemistry and machinery. PCR is a cyclic process of amplifying template DNA through primers, polymerase, deoxynucleoside triphosphates (dNTPs) (nucleotides or building blocks), magnesium ion, buffer, and two to three different incubation temperatures. PCR utilizes (1) a high temperature such as 95°C to convert double-stranded DNA into single-stranded DNA (melting), then (2) a temperature around 55°C to facilitate annealing of a primer pair with target DNA, and (3) a temperature around 70°C to extend annealed primers through polymerase. A typical PCR comprises 30–40 cycles, and it exponentially amplifies target DNA into millions of amplicon copies. PCR is also utilized to amplify RNA. RNA template is first converted into complementary (c)DNA through reverse transcriptase (RT) and then amplified through PCR. In summary, PCR is utilized for qualification, absolute quantitation, and relative quantification of target nucleic acid, and it is used massively in many fields including diagnostics, forensics, and agriculture. PCR applications include detection of pathogens or contaminants, genotyping, relative quantification of gene expression, molecular cloning, sequencing, methylation detection, and site-directed mutagenesis.
Related Knowledge Centers
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