Genotyping by Sequencing for Crop Improvement. Группа авторов

analyzed using bioinformatics tools like stack, PyRAD, dDocent, GATK HaplotypeCaller, GATK unifieldGenotyper, samtools mpileup, etc.

      One enzyme digestion followed by random sharing causes high DNA loss during RAD sequencing. For organisms without a reference genome, a significant portion of the RAD‐Seq data has been discarded due to sequence read errors and the presence of variable sites. To overcome this problem, slight modification in the RAD sequencing protocol has been carried out. Use of two restriction enzymes instead of a single restriction enzyme and removal of the random sharing steps is adopted. The modified method is renamed as double digest RAD (ddRAD).

      Genotyping‐by‐Sequencing (GBS)

      Genotyping‐by‐sequencing (GBS) is also a next‐generation sequencing‐based SNP genotyping method that uses restriction enzymes to reduce the genome complexity. GBS only targets as little as 2.3% of a genome. This reduced representation can be achieved by digestion of genomic DNA with methylation‐sensitive restriction enzymes (REs). Methylation‐sensitive REs do not cut repetitive DNA because of its highly methylated in nature and hence digested fragments generated from low‐copy genomic regions. This helps in avoiding the repetitive regions and targeting low copy regions of the genome (Elshire et al. 2011). In GBS, genomic DNA is extracted and digested with methylation‐sensitive restriction enzymes. Illumina sequencing forward and reverse adaptors ligated to both the end of fragments. The forward adapter is enriched with barcode sequences which is helpful for multiplexing of the samples. Now the PCR amplification is performed using stranded pair‐end Illumina primers. The PCR is used to amplify the GBS library and add the Illumina sequencing regions to the fragment. The prepared GBS library was sequenced using Illumina platform and generated data were analyzed using bioinformatics software’s like Next Generation Sequencing Eclipse Plugin (NGSEP), TASSEL‐GBS, R package msgbsR, etc. (Perea et al. 2016; Wickland et al. 2017).

      2.2.2.2 Allelic Detection

       2.2.2.2.1 Allele‐Specific Primer‐based SNP Genotyping‐Fluidigm

      Fluidigm EP1 is the fast, cost‐effective, and efficient system for medium‐throughput SNP genotyping. This system works on a principle of locus‐specific primer‐based PCR amplification and genotyping (similar to Taqman chemistry). The system incorporates a nanofluidic chip, also known as integrated fluid circuit (IFC) which consists of a network of fluid lines from sample and assay inlet, nanoflex valves, and reaction chambers. This technology has the potential to perform up to 9216 independent TaqMan reactions in a single experiment (Maurice Chan et al. 2011). The nanoflex valve, which is composed of elastomeric rubber, is used to regulate the flow of fluids in the IFC by deflecting under pressure and creating a tight seal is the cornerstone of the IFC.

Figure 2.3 Illustration of various steps involved in the generation of RAD‐based SNP marker generation. (a) Ligate P1 adopter to digested genomic DNA. (b) Poor barcoded sample and shear. (c) Ligate P2 adopter to sheared fragments. (d) selective amplification of RAD tags.

      Source: The figure is reproduced from Baird et al. (2008) available with Creative Commons Attribution License (CC BY).

An IFC is primed in the IFC Controller before loading of samples and assays. The IFC controller pressurizes the control lines and closes the interface valves. After priming of IFC, samples and Taqman assays are loaded into the IFC and then again put into the controller for the mixing of sample and assays into the reaction chambers. In the loading process, on application of pressure, fluid from the sample inlets and assay inlets are pushed into the corresponding fluid lines. The chip utilizes carry over slug design (CS) which allows precise metering and mixing of fluids in a reaction (Unger et al. 2000) chamber (nanolitres of samples and assays are used in a reaction). The loading process takes 45 minutes in the controller and after that, the IFC is being loaded into the standalone PCR and the reaction starts in reaction chambers by thermal cycling in presence of fluorophore‐containing probes. After completion of PCR, the data are acquired in the EP1 fluorescence system and the data analysis is done using bioinformatic tools and Software.

      The processing of plant samples, i.e. DNA requires preamplification, which is done using specific‐target amplification and locus‐specific primers. After diluting the preamplified products with distilled water, PCR amplification with allele‐specific primers is carried out (Kishora et al. 2020). During PCR amplification, two fluorogenic probes corresponding to one of the two alleles in a biallelic SNP are used to probe the SNP site. The probes are made up of a fluorescent reporter dye attached to the probe's 5′ end and a nonfluorescent quencher attached to the probe's 3′ end. The reporter dye's fluorescence is muted when the probe is intact because the reporter dye is close to the quencher. The probe anneals selectively to a complementary sequence during PCR. This causes the probe to cleave, releasing the reporter dye, resulting in increased fluorescence with each PCR cycle. This increase in fluorescence occurs only if the target sequence is complementary to the probe and is amplified during PCR (Seeb et al. 2009).

      MALDI TOF‐based SNP Genotyping‐Sequenom

MALDI MS (matrix‐assisted laser desorption/ionization mass spectrometry) allows for the quick study of high molecular weight biomolecules (e.g. DNA, proteins, peptides, polysaccharides, and other organic compounds) and other chemical substances (e.g. polymers, macromolecules, and hybrid materials). It produces single‐charged ions and has a high detection accuracy and sensitivity. Sequenom incorporates matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) mass spectrometry technique for highly accurate, low‐throughput SNP genotyping. MALDI‐TOF‐based SNP genotyping platforms take the advantage of differences in mass between allele‐specific primer and extension products. During the MALDI process, analyte molecules are mixed with low molecular weight organic molecules like α‐cyano‐4‐hydroxycinnamic acid (CHCA) and 2,5‐dihydroxybenzoic acid (DHB) called matrix and the mixture is crystallized when dried over SpectroCHIP. Then the crystallized matrix‐analyte mixture (a large quantity of matrix is used) is irradiated with a laser beam with an emitting wavelength range in UV or mid IR range. The irradiation causes desorption and ionization of analyte molecules in the form of singly protonated ions. The system uses a TOF mass analyzer. The protonated ions are then accelerated with uniform translational energy on application of the electric field. Then the charged ions are moved to the drift region which is free of electric field. The velocity of charged ions in the drift region is determined by the mass to charge ratio of the ions which in turn is calculated by measuring time required by the ions to travel through the flight tube (Figure 2.4).

Figure 2.4 Schematic illustration of work‐flow in a MALDI‐TOF MS

      Source: The figure is reproduced from Singhal et al., 2015, figure 01(p.03), available with Creative Commons 4.0

      For SNP genotyping, the target regions of the sample (5–10 ng) are amplified for each assay within the multiplex. The PCR product is mixed with the shrimp alkaline phosphatase (SAP) which dephosphorylates any residual dNTPs, which might interfere with the allele‐specific termination at the later stage. The amplification process is followed by a post‐PCR primer extension where an allele‐specific primer is annealed at the adjacent position of the SNP site. The extension reaction cocktail contains four terminating nucleotides (ddNTPs). The extension involves the addition of a single nucleotide and hence alleles are differentiated by