SNP Discovery through Next- Generation Sequencing and Its Applications. Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada R3. T 2. N2. 2Department of Applied Genomics, Vineland Research and Innovation Centre, Vineland Station, ON, Canada L0. R 2. E0. 3Cereal Research Centre, Agriculture and Agri- Food Canada, Winnipeg, MB, Canada R3. T 2. M9. Copyright В© 2. Santosh Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The decreasing cost along with rapid progress in next- generation sequencing and related bioinformatics computing resources has facilitated large- scale discovery of SNPs in various model and nonmodel plant species. Large numbers and genome- wide availability of SNPs make them the marker of choice in partially or completely sequenced genomes. Although excellent reviews have been published on next- generation sequencing, its associated bioinformatics challenges, and the applications of SNPs in genetic studies, a comprehensive review connecting these three intertwined research areas is needed. This paper touches upon various aspects of SNP discovery, highlighting key points in availability and selection of appropriate sequencing platforms, bioinformatics pipelines, SNP filtering criteria, and applications of SNPs in genetic analyses. The use of next- generation sequencing methodologies in many non- model crops leading to discovery and implementation of SNPs in various genetic studies is discussed. Development and improvement of bioinformatics software that are open source and freely available have accelerated the SNP discovery while reducing the associated cost. Key considerations for SNP filtering and associated pipelines are discussed in specific topics. Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G A (rs17822931), in the ABCC11 gene is responsible for.
A list of commonly used software and their sources is compiled for easy access and reference. Introduction Molecular markers are widely used in plant genetic research and breeding. Single Nucleotide Polymorphisms (SNPs) are currently the marker of choice due to their large numbers in virtually all populations of individuals. The applications of SNP markers have clearly been demonstrated in human genomics where complete sequencing of the human genome led to the discovery of several million SNPs [1] and technologies to analyze large sets of SNPs (up to 1 million) have been developed. SNPs have been applied in areas as diverse as human forensics [2] and diagnostics [3], aquaculture [4], marker assisted- breeding of dairy cattle [5], crop improvement [6], conservation [7], and resource management in fisheries [8]. Functional genomic studies have capitalized upon SNPs located within regulatory genes, transcripts, and Expressed Sequence Tags (ESTs) [9, 1. Until recently large scale SNP discovery in plants was limited to maize, Arabidopsis, and rice [1. LASERJET PRO 300 COLOR MFP M375nw HP LASERJET PRO 300 COLOR MFP M375nw Product Number: CE903A ENERGY STAR® qualified models; see http:// 1Measured using ISO/IEC 24734, excludes first set of test. Alexander Elliot Anderson 'Alex' Salmond (/ ˈ s æ m ən d /; born 31 December 1954) is a Scottish politician who served as the fourth First Minister of Scotland from 2007 to 2014. He was the leader of the Scottish National. Genetic applications such as linkage mapping, population structure, association studies, map- based cloning, marker- assisted plant breeding, and functional genomics continue to be enabled by access to large collections of SNPs. Arabidopsis thaliana was the first plant genome sequenced [1. In the year 2. 01. With the ever increasing throughput of next- generation sequencing (NGS), de novo and reference- based SNP discovery and application are now feasible for numerous plant species. Sequencing refers to the identification of the nucleotides in a polymer of nucleic acids, whether DNA or RNA. Since its inception in 1. Tremendous improvements in sequencing have led to the generation of large amounts of DNA information in a very short period of time [1. The analyses of large volumes of data generated through various NGS platforms require powerful computers and complex algorithms and have led to a recent expansion of the bioinformatics field of research. This book chapter focuses on the a priori discovery of SNPs through NGS, bioinformatics tools and resources, and the various downstream applications of SNPs. History and Evolution of Sequencing Technologies. Invention of Sequencing. In 1. 97. 7, two sequencing methods were developed and published. The Sanger method is a sequencing- by- synthesis (SBS) method that relies on a combination of deoxy- and dideoxy- labeled chain terminator nucleotides [2. The first complete genome sequencing, that of bacteriophage phi X1. The chemical modification followed by cleavage at specific sites method also published in 1. In contrast, the Sanger method, for which Frederick Sanger was awarded his second Nobel Prize in chemistry in 1. Sequencing Technologies. In the last decade, new sequencing technologies have outperformed Sanger- based sequencing in throughput and overall cost, if not quite in sequence length and error rate [2. This section will focus on the three main NGS platforms as well as the two main third- generation sequencing (TGS) platforms, their throughput and relative cost. We made every effort to ensure the accuracy of the data at the time of submission. However, the cost and throughput of these sequencing platforms change rapidly and, as such, our analysis only represents a snapshot in time. The flux of innovation in this field imposes a need for constant assessment of the technologies’ potentials and realignment of research goals. Roche (4. 54) Sequencing. Pyrosequencing was the first of the new highly parallel sequencing technologies to reach the market [2. It is commonly referred to as 4. HP LaserJet Pro 400 Color MFP M475 Series HP LaserJet Pro 400 Color MFP M475 Series Product Number: CE863A and CE864A ENERGY STAR® qualified models; see www.hp.com/go/energystar 1Measured using ISO/IEC 24734, excludes first. 2013 sourcecode technology holdings, inc. all rights reserved. sourcecode software products are protected by one or more u.s. patents. other patents pending. sourcecode, k2, the four squares logo, k2 blackpearl, k2 connect and. Page 1 Version 1.14 November 24, 2010 837 Health Care Claim Companion Guide Professional and Institutional Version 1.14 November 24, 2010. It is an SBS method where single fragments of DNA are hybridized to a capture bead array and the beads are emulsified with regents necessary to PCR amplifying the individually bound template. Each bead in the emulsion acts as an independent PCR where millions of copies of the original template are produced and bound to the capture beads which then serve as the templates for the subsequent sequencing reaction. The individual beads are deposited into a picotiter plate along with DNA polymerase, primers, and the enzymes necessary to create fluorescence through the consumption of inorganic phosphate produced during sequencing. The instrument washes the picotiter plate with each of the DNA bases in turn. As template- specific incorporation of a base by DNA polymerase occurs, a pyrophosphate (PPi) is produced. This pyrophosphate is detected by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) through the generation of a light signal following the conversion of PPi into ATP [2. Thus, the wells in which the current nucleotides are being incorporated by the sequencing reaction occurring on the bead emit a light signal proportional to the number of nucleotides incorporated, whereas wells in which the nucleotides are not being incorporated do not. The instrument repeats the sequential nucleotide wash cycle hundreds of times to lengthen the sequences. The 4. 54 GS FLX Titanium XL+ platform currently generates up to 7. MB of raw 7. 50 bp reads in a 2. The technology has difficulty quantifying homopolymers resulting in insertions/deletions and has an overall error rate of approximately 1%. Reagent costs are approximately $6,2. Illumina Sequencing. Illumina technology, acquired by Illumina from Solexa, followed the release of 4. With this sequencing approach, fragments of DNA are hybridized to a solid substrate called a flow cell. In a process called bridge amplification, the bound DNA template fragments are amplified in an isothermal reaction where copies of the template are created in close proximity to the original. This results in clusters of DNA fragments on the flow cell creating a “lawn” of bound single strand DNA molecules. The molecules are sequenced by flooding the flow cell with a new class of cleavable fluorescent nucleotides and the reagents necessary for DNA polymerization [2. A complementary strand of each template is synthesized one base at a time using fluorescently labeled nucleotides. The fluorescent molecule is excited by a laser and emits light, the colour of which is different for each of the four bases. The fluorescent label is then cleaved off and a new round of polymerization occurs. Unlike 4. 54 sequencing, all four bases are present for the polymerization step and only a single molecule is incorporated per cycle. The flagship Hi. Seq. Illumina can generate up to 6. GB per run with a read length of 1. The Illumina technique can generate sequence from opposite ends of a DNA fragment, so called paired- end (PE) reads. Reagent costs are approximately $2. Applied Biosystems (SOLi. D) Sequencing. The SOLi. D system was jointly developed by the Harvard Medical School and the Howard Hughes Medical Institute [2. The library preparation in SOLi. D is very similar to Roche/4. DNA template, beads, primers, and PCR components. Beads that contain PCR products amplified by emulsion PCR are enriched by a proprietary process. The DNA templates on the beads are modified at their 3′ end to allow attachment to glass slides. A primer is annealed to an adapter on the DNA template and a mixture of fluorescently tagged oligonucleotides is pumped into the flow cell. When the oligonucleotide matches the template sequence, it is ligated onto the primer and the unincorporated nucleotides are washed away. A charged couple device (CCD) camera captures the different colours attached to the primer. Each fluorescence wavelength corresponds to a particular dinucleotide combination. After image capture, the fluorescent tag is removed and new set of oligonucleotides are injected into the flow cell to begin the next round of DNA ligation [1. This sequencing- by- ligation method in SOLi. D- 5. 50. 0x. 1 platform generates up to 1,4. PE reads of nt each with an error rate of 0. Although widely accepted and used, the NGS platforms suffer from amplification biases introduced by PCR and dephasing due to varying extension of templates.
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