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.
The Gene for t-PA
Cornelis Kluft in Tissue-Type Plasminogen Activator (t-PA): Physiological and Clinical Aspects, 1988
The general strategy for the construction of a genomic library first requires the isolation of DNA fragments from a donor organism. This is done by either shearing or enzymatically cleaving the chromosomal DNA. Using the enzyme DNA ligase, genomic DNA fragments are joined to a suitable cloning vector in vitro. The recombinant DNA molecules are then introduced into E. coli cells where they multiply (Figure 1). When a specific gene is isolated from a genomic library, it will be in the form found on the chromosome. It will therefore contain exons, introns, and regulatory regions, such as promotor and enhancer sequences. Since a gene isolated from a genomic library contains regulatory sequences, it can be reintroduced into eucaryotic cells, and its expression studied.
Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease
Jinghua Hu, Yong Yu in Polycystic Kidney Disease, 2019
The hybridization capture-based target enrichment approach employs oligonucleotide probes to capture target sequences in an NGS library.31 The principle is based on the hybridization of prepared DNA fragments complementary to the regions of interest to a standard shotgun sequencing library made from genomic DNA. Hybridization of the target regions can occur either on a solid surface or in solution. Solid-phase methods use a microarray, where the complementary probes are affixed to a glass slide for the hybrid capture reaction. In the solution-based or fluid-phase method, prior to NGS, pools of biotinylated oligonucleotide probes are hybridized to a sequencing library in solution. Following hybridization, the biotinylated probes are pulled down using streptavidin-coated magnetic beads to achieve libraries highly enriched for the target regions, and nontarget sequences are washed away. There is also the possibility to incorporate molecular barcodes in the DNA library, either to multiplex multiple samples in the same experiment, or to improve sequencing error detection and improve base calling accuracy for variants with a low variant allele frequency (i.e., somatic variants).32 The amount of DNA required for library preparation varies based on different protocols and kits. Usually 1–3 μg DNA input is needed but specific kits designed for samples with low availability, enable libraries even from 10 ng DNA input. Hybrid capture-based target enrichment is commercialized mostly by Agilent Technologies (SureSelect) and Roche-NimbleGen (SeqCap/SeqCap EZ), and they offer products including library preparation and target enrichment kits, catalog and custom probes, software solutions, sample quality control, and automation platforms.
The gut microbiome and metabolome in kidney transplant recipients with normal and moderately decreased kidney function
Published in Renal Failure, 2023
Yang Lan, Duo Wang, Jiayang He, Hongji Yang, Yifu Hou, Wenjia Di, Hailian Wang, Xiangwei Luo, Liang Wei
Construction of DNA Library. The DNA library was constructed according to the Novogene Co., Ltd (Beijing, China) manufacturer’s instructions. NEBNext Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) was used to generate sequencing libraries. Index codes were added to each sample to identify attributes for each sequence. The qualified DNA samples were randomly sheared to a size of 350 bp by sonication. Then these fragments were end-repaired, ligated with Illumina sequencing adapters, and PCR amplified. AMPure XP system (Beckman Coulter, CA, USA) was used to purify the PCR products, Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the size distribution of DNA library and real-time PCR was used to quantify DNA libraries [15].
The prenatal exome – a door to prenatal diagnostics?
Published in Expert Review of Molecular Diagnostics, 2021
James S Castleman, Elizabeth Wall, Stephanie Allen, Denise Williams, Samantha Doyle, Mark D Kilby
NGS refers to a diverse group of high-throughput sequencing technologies developed over the last decade [9] although they share the same basic steps. It can be used to sequence single genes, gene panels (multiple genes), exomes, or whole genomes [9]. After basic checks to confirm identity and rule out contamination, genomic DNA (gDNA) is fragmented into a library. Regions of interest (e.g., a gene panel or clinical exome) are captured and amplified in an optional target enrichment step. Massively parallel sequencing detects and produces millions of reads. Bioinformatics software aligns the reads to a reference genome and carries out quality control checks. One of the most common NGS technologies currently used in medical genetics is the ‘sequencing-by-synthesis’ technique [9], and this technology has largely been integrated into National Health Service (NHS) laboratories (in the United Kingdom) to embed genome sequencing technologies in mainstream clinical services. DNA samples are pooled in one sequencing reaction by attaching a molecular barcode unique to each sample to every DNA fragment, increasing throughput and cost-effectiveness [10].
Effects of spaceflight on the composition and function of the human gut microbiota
Published in Gut Microbes, 2020
Zizhong Liu, Gui Luo, Ruikai Du, Weijia Sun, Jianwei Li, Haiyun Lan, Pu Chen, Xinxin Yuan, Dengchao Cao, Yuheng Li, Caizhi Liu, Shuai Liang, Xiaoyan Jin, Ruifu Yang, Yujing Bi, Yanping Han, Ping Cao, Wei Zhao, Shukuan Ling, Yingxian Li
Stool samples (180–220 mg) were weighed in 2-ml microcentrifuge tubes and placed on ice. Total DNA from the fecal microbiotas was extracted using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany) per the manufacturer’s instructions (see the QIAamp Fast DNA Stool Mini Kit Handbook, www.qiagen.com/handbooks). The degradation degree and potential contamination of the DNA were analyzed using 1% agarose gels. The DNA purity was determined using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), and the DNA concentration was measured using the Qubit® dsDNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). One microgram of qualified DNA was used to construct the library. DNA samples were fragmented to 350 bp by sonication, then the DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. Libraries were analyzed for size distribution using the Agilent2100 Bioanalyzer (Agilent, USA) and quantified via real-time PCR. The libraries were sequenced on an Illumina HiSeq platform.
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