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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.
The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Sequencing of nearly an entire human genome was first accomplished in 2000, partly through the use of Shotgun Sequencing technology. While full genome Shotgun Sequencing for small (i.e., 4,000–7,000 base pairs) genomes was already in use in 1979, broader applications followed based on Pairwise End Sequencing (also known as “Double-Barrel Shotgun” sequencing). Then, as sequencing projects began to take on longer and more complicated genomes, it was realized that useful information could be obtained by sequencing from both ends of a DNA fragment. Although keeping track of the paired data was more cumbersome than standard sequencing from a single end of two distinct fragments, this approach allowed in reconstruction of the sequence of the original target fragment.
Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Matthew Lanktree, Amirreza Haghighi, Xueweng Song, York Pei
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.
Microbiome distinctions between the CRC carcinogenic pathways
Published in Gut Microbes, 2021
Lauren DeDecker, Bretton Coppedge, Julio Avelar-Barragan, William Karnes, Katrine Whiteson
A fifth limitation is that some studies have been limited in the type of sequencing used to characterize the microbiome. The prevailing approach is 16S rRNA amplicon sequencing, which focuses on an individual, universal marker gene. 16S rRNA gene sequencing can elucidate which bacteria are present in a sample but does not include information about specific metabolic capacities of individual strains or species. The increased resolution provided by whole community shotgun metagenomic sequencing may in part explain why a recent meta-analysis of 16S rRNA fecal microbiomes from CRC patients failed to find biomarkers of CRC,45 while subsequent shotgun sequencing meta-analyses detected increased protein and mucin catabolism genes and reduced carbohydrate degradation genes.46 Thus, we recommend that future studies consider using shotgun sequencing for their sample analysis. In addition, it is critical that microbiome researchers use consistent standards, such as having positive and negative controls as part of every study and using spike-in standards to get semi-quantitative data.47 It is also beneficial to use consistent taxonomic resolution. Research data and analyses should be open access to support rigorous and reproducible science.
Complex interactions between the microbiome and cancer immune therapy
Published in Critical Reviews in Clinical Laboratory Sciences, 2019
Drew J. Schwartz, Olivia N. Rebeck, Gautam Dantas
After the sequencing data is obtained, the first question asked is ‘who is there?’ Both 16S rRNA and shotgun metagenomic sequencing can answer this question (Figure 1(C)). Data from sequencing analysis can be visualized in terms of relative abundance of taxa within a sample or as a similarity or dissimilarity ordination plot based on measures of alpha and beta diversity (Figure 1(C)). Alpha diversity measurements describe intra-sample diversity: measurements include Shannon diversity and species richness. Beta diversity measures are pairwise comparisons of dissimilarity between all sample pairs and are typically viewed as an ordination plot with principal coordinate analysis (PCoA). Measures of beta diversity include Bray-Curtis, binary-Jaccard, and weighted UniFrac (Figure 1(C)) (reviewed extensively in [44]). In addition to overall assessments of similarity and dissimilarity between samples, these same measures can be used to examine differences in microbial gene content (e.g. functional pathways and resistance genes) from metagenomic shotgun sequencing (Figure 1(C)) (reviewed in [27]). Although shotgun metagenomic sequencing is more expensive than 16S rRNA gene sequencing per sample when performed at a depth of 2.5 – 5 million reads per sample, the validation of shallow shotgun sequencing with as few as 500,000 reads per sample increases the affordability of this method for large datasets [33]. Thus, in designing any study interrogating the microbiome, it is vital to determine the desired downstream analyses so that sampling schema and sequencing depth can be planned appropriately.
The Ocular Microbiome: Molecular Characterisation of a Unique and Low Microbial Environment
Published in Current Eye Research, 2019
All studies to date have focused their investigation on the bacterial inhabitants on the ocular surface but have yet to probe deeply into the larger biosphere of the virome and mycobiome. One study has used metagenomic sequencing and found that approximately 98% of sequencing reads were of bacterial origin, while reads of viral and fungal origin accounted for approximately 1% each.9 Another study assessed for the presence of torque teno virus on the healthy ocular surface and found that 65% of subject samples had a positive signal for torque teno virus,10 which is associated with culture-negative endophthalmitis and seasonal hyperacute panuveitis.73 Surveying for different viruses using whole sample shotgun sequencing represents a greater challenge and increased costs. In terms of the fungal community, culture-based studies have shown that fungi are isolated sporadically from the ocular surface in healthy eye (4%), whereas culture-independent studies using panfungal PCR primers tested positive for fungal DNA more frequently (23%).74 Based on recent findings, it has been proposed that bacteriophage adherence to mucosal surfaces benefits the host by limiting mucosal bacteria providing non-host-derived antimicrobial defence.75 Very little is currently known of the role of bacteriophage, which significantly outnumber even bacteria and colonise all niches of the human body,76 and it will be interesting to see what role these non-bacterial microorganisms have on eye health and disease.