Restriction site associated DNA markers

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Genomic DNA is first digested with a specific restriction enzyme(s) to fragment the DNA. For restriction fragment length polymorphism (RFLP) analysis, these fragments are then visualized by gel electrophoresis. For RADseq, restriction fragments are ligated to an adapter that makes them readable by sequencing machines (not pictured), then fragments of a selected size range are sequenced using next-generation sequencing methods, aligned, and compared. RADseq schematic.pdf
Genomic DNA is first digested with a specific restriction enzyme(s) to fragment the DNA. For restriction fragment length polymorphism (RFLP) analysis, these fragments are then visualized by gel electrophoresis. For RADseq, restriction fragments are ligated to an adapter that makes them readable by sequencing machines (not pictured), then fragments of a selected size range are sequenced using next-generation sequencing methods, aligned, and compared.

Restriction site associated DNA (RAD) markers are a type of genetic marker which are useful for association mapping, QTL-mapping, population genetics, ecological genetics and evolutionary genetics. The use of RAD markers for genetic mapping is often called RAD mapping. An important aspect of RAD markers and mapping is the process of isolating RAD tags, which are the DNA sequences that immediately flank each instance of a particular restriction site of a restriction enzyme throughout the genome. [1] Once RAD tags have been isolated, they can be used to identify and genotype DNA sequence polymorphisms mainly in form of single nucleotide polymorphisms (SNPs). [1] Polymorphisms that are identified and genotyped by isolating and analyzing RAD tags are referred to as RAD markers. Although genotyping by sequencing presents an approach similar to the RAD-seq method, they differ in some substantial ways. [2] [3] [4]

Contents

Isolation of RAD tags

The use of the flanking DNA sequences around each restriction site is an important aspect of RAD tags. [1] The density of RAD tags in a genome depends on the restriction enzyme used during the isolation process. [5] There are other restriction site marker techniques, like RFLP or amplified fragment length polymorphism (AFLP), which use fragment length polymorphism caused by different restriction sites, for the distinction of genetic polymorphism. The use of the flanking DNA-sequences in RAD tag techniques is referred as reduced-representation method. [2]

The initial procedure to isolate RAD tags involved digesting DNA with a particular restriction enzyme, ligating biotinylated adapters to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, and isolating the biotinylated fragments using streptavidin beads. [1] This procedure was used initially to isolate RAD tags for microarray analysis. [1] [6] [7] More recently, the RAD tag isolation procedure has been modified for use with high-throughput sequencing on the Illumina platform, which has the benefit of greatly reduced raw error rates and high throughput. [5] The new procedure involves digesting DNA with a particular restriction enzyme (for example: SbfI, NsiI,…), ligating the first adapter, called P1, to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, preparing the sheared ends into blunt ends and ligating the second adapter (P2), and using PCR to specifically amplify fragments that contain both adapters. Importantly, the first adapter contains a short DNA sequence barcode, called MID (molecular identifier) that is used as a marker to identify different DNA samples that are pooled together and sequenced in the same reaction. [5] [8] The use of high-throughput sequencing to analyze RAD tags can be classified as reduced-representation sequencing, which includes, among other things, RADSeq (RAD-Sequencing). [2]

Detection and genotyping of RAD markers

Once RAD tags have been isolated, they can be used to identify and genotype DNA sequence polymorphisms such as single nucleotide polymorphisms (SNPs). [1] [5] These polymorphic sites are referred to as RAD markers. The most efficient way to find RAD tags is by high-throughput DNA sequencing, [5] [8] called RAD tag sequencing, RAD sequencing, RAD-Seq, or RADSeq.

Prior to the development of high-throughput sequencing technologies, RAD markers were identified by hybridizing RAD tags to microarrays. [1] [6] [7] Due to the low sensitivity of microarrays, this approach can only detect either DNA sequence polymorphisms that disrupt restriction sites and lead to the absence of RAD tags or substantial DNA sequence polymorphisms that disrupt RAD tag hybridization. Therefore, the genetic marker density that can be achieved with microarrays is much lower than what is possible with high-throughput DNA-sequencing. [9]

History

RAD markers were first implemented using microarrays and later adapted for NGS (Next-Generation-Sequencing). [9] It was developed jointly by Eric Johnson and William Cresko's laboratories at the University of Oregon around 2006. They confirmed the utility of RAD markers by identifying recombination breakpoints in D. melanogaster and by detecting QTLs in threespine sticklebacks. [1]

ddRADseq

In 2012 a modified RAD tagging method called double digest RADseq (ddRADseq) was suggested. [10] [11] By adding a second restriction enzyme, replacing the random shearing, and a tight DNA size selection step it is possible to perform low-cost population genotyping. This can be an especially powerful tool for whole-genome scans for selection and population differentiation or population adaptation. [11]

hyRAD

A study in 2016 presented a novel method called hybridization RAD (hyRAD), [12] where biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. DNA fragments are first generated using ddRADseq protocol applied to fresh samples, and used as hybridization-capture probes to enrich shotgun libraries in the fragments of interest. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museomics. Another advantage of the method is not relying on the restriction site presence, improving among-sample loci coverage. The technique was first tested on museum and fresh samples of Oedaleus decorus , a Palearctic grasshopper species, and later implemented in regent honeyeater, [13] arthropods, [14] among other species. A lab protocol was developed to implement hyRAD in birds. [15]

See also

Related Research Articles

In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, populations, or species or to pinpoint the locations of genes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

<span class="mw-page-title-main">Single-nucleotide polymorphism</span> Single nucleotide in genomic DNA at which different sequence alternatives exist

In genetics and bioinformatics, a single-nucleotide polymorphism is a germline substitution of a single nucleotide at a specific position in the genome that is present in a sufficiently large fraction of considered population.

<span class="mw-page-title-main">Serial analysis of gene expression</span> Molecular biology technique

Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGE and the most recent SuperSAGE. Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.

A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change, or a long one, like minisatellites.

<span class="mw-page-title-main">Gene mapping</span> Process of locating specific genes

Gene mapping or genome mapping describes the methods used to identify the location of a gene on a chromosome and the distances between genes. Gene mapping can also describe the distances between different sites within a gene.

<span class="mw-page-title-main">Ancestry-informative marker</span>

In population genetics, an ancestry-informative marker (AIM) is a single-nucleotide polymorphism that exhibits substantially different frequencies between different populations. A set of many AIMs can be used to estimate the proportion of ancestry of an individual derived from each population.

Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents. Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual.

In molecular biology, SNP array is a type of DNA microarray which is used to detect polymorphisms within a population. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome. Around 335 million SNPs have been identified in the human genome, 15 million of which are present at frequencies of 1% or higher across different populations worldwide.

SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.

<span class="mw-page-title-main">Bisulfite sequencing</span> Lab procedure detecting 5-methylcytosines in DNA

Bisulfitesequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.

The following outline is provided as an overview of and topical guide to genetics:

Population genomics is the large-scale comparison of DNA sequences of populations. Population genomics is a neologism that is associated with population genetics. Population genomics studies genome-wide effects to improve our understanding of microevolution so that we may learn the phylogenetic history and demography of a population.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

Methylated DNA immunoprecipitation is a large-scale purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. et al. in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type.

Diversity Arrays Technology (DArT) is a high-throughput genetic marker technique that can detect allelic variations to provides comprehensive genome coverage without any DNA sequence information for genotyping and other genetic analysis. The general steps involve reducing the complexity of the genomic DNA with specific restriction enzymes, choosing diverse fragments to serve as representations for the parent genomes, amplify via polymerase chain reaction (PCR), insert fragments into a vector to be placed as probes within a microarray, then fluorescent targets from a reference sequence will be allowed to hybridize with probes and put through an imaging system. The objective is to identify and quantify various forms of DNA polymorphism within genomic DNA of sampled species.

Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterations and characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously.

<span class="mw-page-title-main">Exome sequencing</span> Sequencing of all the exons of a genome

Exome sequencing, also known as whole exome sequencing (WES), is a genomic technique for sequencing all of the protein-coding regions of genes in a genome. It consists of two steps: the first step is to select only the subset of DNA that encodes proteins. These regions are known as exons—humans have about 180,000 exons, constituting about 1% of the human genome, or approximately 30 million base pairs. The second step is to sequence the exonic DNA using any high-throughput DNA sequencing technology.

Disease gene identification is a process by which scientists identify the mutant genotypes responsible for an inherited genetic disorder. Mutations in these genes can include single nucleotide substitutions, single nucleotide additions/deletions, deletion of the entire gene, and other genetic abnormalities.

In the field of genetic sequencing, genotyping by sequencing, also called GBS, is a method to discover single nucleotide polymorphisms (SNP) in order to perform genotyping studies, such as genome-wide association studies (GWAS). GBS uses restriction enzymes to reduce genome complexity and genotype multiple DNA samples. After digestion, PCR is performed to increase fragments pool and then GBS libraries are sequenced using next generation sequencing technologies, usually resulting in about 100bp single-end reads. It is relatively inexpensive and has been used in plant breeding. Although GBS presents an approach similar to restriction-site-associated DNA sequencing (RAD-seq) method, they differ in some substantial ways.

Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst non-coding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. Transcriptomics technologies provide a broad account of which cellular processes are active and which are dormant. A major challenge in molecular biology is to understand how a single genome gives rise to a variety of cells. Another is how gene expression is regulated.

References

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  3. ↑ Campbell EO, Brunet BM, Dupuis JR, Sperling FA (2018). "Would an RRS by any other name sound as RAD?". Methods in Ecology and Evolution. 9 (9): 1920–1927. doi: 10.1111/2041-210X.13038 .
  4. ↑ Vaux F, Dutoit L, Fraser CI, Waters JM (2022). "Genotyping-by-sequencing for biogeography". Journal of Biogeography. 50 (2): 262–281. doi: 10.1111/jbi.14516 .
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  8. 1 2 Hohenlohe PA, Bassham S, Etter PD, Stiffler N, Johnson EA, Cresko WA (February 2010). "Population genomics of parallel adaptation in threespine stickleback using sequenced RAD tags". PLOS Genetics. 6 (2): e1000862. doi: 10.1371/journal.pgen.1000862 . PMC   2829049 . PMID   20195501.
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  10. ↑ Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012). "Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species". PLOS ONE. 7 (5): e37135. Bibcode:2012PLoSO...737135P. doi: 10.1371/journal.pone.0037135 . PMC   3365034 . PMID   22675423.
  11. 1 2 Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012). "Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species". PLOS ONE. 7 (5): e37135. Bibcode:2012PLoSO...737135P. doi: 10.1371/journal.pone.0037135 . PMC   3365034 . PMID   22675423.
  12. ↑ Suchan T, Pitteloud C, Gerasimova NS, Kostikova A, Schmid S, Arrigo N, et al. (2016). Orlando L (ed.). "Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens". PLOS ONE. 11 (3): e0151651. Bibcode:2016PLoSO..1151651S. doi: 10.1371/journal.pone.0151651 . PMC   4801390 . PMID   26999359.
  13. ↑ Crates R, Olah G, Adamski M, Aitken N, Banks S, Ingwersen D, et al. (2019). Russello MA (ed.). "Genomic impact of severe population decline in a nomadic songbird". PLOS ONE. 14 (10): e0223953. Bibcode:2019PLoSO..1423953C. doi: 10.1371/journal.pone.0223953 . PMC   6812763 . PMID   31647830.
  14. ↑ Faircloth, Brant C. (2017). Gilbert, M. (ed.). "Identifying conserved genomic elements and designing universal bait sets to enrich them". Methods in Ecology and Evolution. 8 (9): 1103–1112. doi: 10.1111/2041-210X.12754 . ISSN   2041-210X. S2CID   51896564.
  15. ↑ Olah G, Aitken N, Suchan T (2018-01-25). "hyRAD for birds v1". Protocols.io. doi:10.17504/protocols.io.mt2c6qe.
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