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Review
. 2013 Jun;62(6):920-32.
doi: 10.1136/gutjnl-2011-301935. Epub 2012 May 1.

Next generation sequencing and a new era of medicine

Graham Casey  1 , David Conti, Robert Haile, David Duggan
Affiliations
Review

Next generation sequencing and a new era of medicine

Graham Casey et al. Gut. 2013 Jun.
No abstract available

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Figures

Figure 1
Figure 1. Overview of the next generation DNA sequencing workflow
Nucleic acid sources from blood, tissue, and cell culture are all compatible with next generation sequencing (NGS). Nucleic acids including DNA and RNA from germ-line as well as somatic sources can be used in the NGS assays. Depending on the choice of experimental design, samples can be enriched for specific targets (e.g., the complete coding sequence of the human genome or exome, individual gene(s), or other custom needs); RNA specimens in most cases are converted to ds-cDNA at this time (exception being direct sequencing of RNA molecules on the Heliscope). Following fragmentation, if needed, nucleic acid samples are end repaired, A-tailed, linker-modified, and, in most cases, amplified prior to sequencing. Linker-modified and amplified nucleic acids can be sequenced using any number of next generation sequencing approaches depending on the needs of the study design. Raw sequence data is processed via a stabilizing but still dynamic pipeline; the highlights of the procedure are identified here. Numerous public and commercially available programs are used for variant calling, data visualization, and annotation. Finally, annotation of the identified variants along with other analyses can be used to improve the understanding of the genetic basis of disease. Of course, additional studies will likely be required for a complete understanding of the functional consequences of the identified variant(s). Specific details on all of the processes depicted here can be found in the references referred to in the text as well as references contained within. *Depending on the RNA isolation protocol(s), total RNA, mRNA, miRNA, lncRNAs can be studied.
Figure 2
Figure 2. Solid phase amplification used by Illumina sequencers
In solid phase amplification forward and reverse primers are covalently attached to a support at high density. Adaptors complementary to these primers are ligated to DNA fragments, the adapter modified DNA fragment is then hybridized to the immobilized primers, and amplified in situ following looping and annealing of the free end of the DNA fragment to a complementary primer on the surface. Repeated denaturation and primer extension results in localized amplification of single molecules in clusters across the surface of the slide.
Figure 3
Figure 3. Emulsion PCR used by 454 and Life Technologies, for example
In emulsion PCR, adapters containing universal sequencing primers are ligated to the DNA fragments allowing a complex genome to be PCR amplified using a common set of PCR primers. Prior to the emulsion PCR, fragments are separated into single strands and captured onto beads under conditions favoring capture of single DNA molecules on a single bead, and following PCR amplification, several thousands of copies of the same fragment are generated. The spatially separated adapter modified templates are then physically bound to a solid support for NGS where the repeated cycling and reverse incorporation of nucleotides and image capture of hundreds of millions of templates is accomplished in parallel.
Figure 4
Figure 4
Overview of the core TGFβ pathway. TGFβ gene regions identified through GWAS include, BMP2, BMP4, SMAD7, GREM1, LAMA5 and RHPN2
See this image and copyright information in PMC

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