Genomics in medicine: A new era in medicine

Table 1 Characteristics of commonly used genomic tools

Tools for genomics	Principle of use	Pros and application	Limitation
Genome-wide association studies (GWAS)	Gene mapping study using DNA microarray to identify the association between SNP and specific risk alleles that are more prevalent in cases than in controls, via linkage disequilibrium	Has potential for population-based application. Example — The Severe COVID-19 GWAS Group[34] studied patients with respiratory failure from severe COVID-19 and narrowed down the genetic susceptibility locus to a gene cluster on chromosome locus 3p21.31. They also verified the potential involvement of the ABO blood group system	Does not establish causality but only an association with SNP; Missing heritability- cannot explain variance in complex traits or genes with a small effect size; Does not account for epigenetic changes and epistasis (gene-gene interaction); GWAS data catalog mostly from individuals of European descent which may limit application in minority population[35]
Expression quantitative trait loci (eQTL) analysis	Links SNPs to changes in gene expression by measuring the expression of many genes simultaneously in microarrays. Helps to narrow down to SNPs more likely to impact the disease condition	Provides better insight into specific causal mechanisms[36]; Liver eQTL — useful in pharmacogenomic studies by analyzing Epistatic eQTL Interactions[37]	Limited tissue interrogation will give misleading biological interpretations about the gene mediating the regulatory effect to increase disease risk[38]
Deep sequencing or Next-generation sequencing	Exome sequencing: 85% of known disease-causing mutations in Mendelian disorders are found in exons. Exome sequencing is a useful tool to find the causal genes for Mendelian disorders	Reduced cost and limited data to interpret; Linkage study design is unsuitable for extremely rare and sporadic Mendelian disorders for which exome sequencing would be more practical[39]	Exome sequencing: It can miss pathogenic variants in a non-coding region. Repetitive regions (e.g., pseudogenes) can confound results in whole-exome sequencing[41]; Potentiate technical biases regarding exon capture limiting its use in detecting copy-number variants as well as in genomic regions where capture is less efficient[42]
	Whole-genome sequencing: Can sequence every nucleotide base in the human genome (approximately 3.3 × 109 base pairs)	Whole-genome sequencing: Avoids inherent biases of exome capture	Whole-genome sequencing: Too much data but little clinical knowledge available to interpret; Higher cost compared to clinical utility
	Targeted gene panel: Provides information on prespecified disease-associated genes	Examples: Rapid whole-genome sequencing to investigate extensively drug-resistant (XDR) tuberculosis[40]
RNA-seq	Uses NGS to analyze RNA expression patterns or transcriptome profiling by reverse transcription of RNA sample to complementary DNAs (cDNA) and PCR amplification	Can be used: to analyze RNA expression profile at single cell level or quantify gene expression[43]; to obtain data on novel transcripts and is not limited by availability of reference genome data[44]; to identify alternatively spliced genes; to detect allele-specific gene expression[44]	cDNA synthesis and PCR amplification steps can introduce bias and errors[44]
Epigenomics	Epigenomics involves methods used to identify DNA methylation and histone modifications. Sodium bisulfite can identify unmethylated cytosines due to its ability to convert unmethylated cytosines to uracil. However the methylated cytosine is resistant to this conversion. Methylation-dependent restriction enzymes are used for DNA methylation analysis[45]. Chromatin immunoprecipitation (ChIP) is used for the investigation of histone modifications	ChIP allows precise mapping of the DNA-protein interaction in living cells. Cross-linked protein-DNA complex can be treated with exonucleases to remove cross-linked DNA sequences that are not avidly bound to protein of interest. This is called ChIP-Exo. This allows mapping of in vivo protein occupancy at single nucleotide-level resolution[47]	Needs design of antibodies specific to DNA-bound protein of interest which could be modified histone or transcription factors
	Immunoprecipitation techniques: ChIP on Chip; ChIP-Seq. Chromatin is isolated from the sample and the DNA involved in DNA protein cross-linked complex is isolated using antibodies specific to the DNA-bound protein. The isolated DNA is amplified using PCR and analyzed using gel electrophoresis imaging, microarray hybridization (ChIP-chip), or direct sequencing with NGS (ChIP-Seq)[46]
Transcriptomics	Northern blot: RNA molecules separated by gel electrophoresis by size and subsequently hybridized with labeled complementary ssDNA and detected using chemic luminescence or autoradiography	Northern blot can both quantify the amount of RNA and also determine the size of mRNA transcript. Can detect transcript variant of genes[49]	Northern blot-need radioactive probes and has lower sensitivity
	Ribonuclease (RNase) protection assay: Differs from northern blot by use of antisense RNA probes called riboprobes	RNase protection assay: It can simultaneously detect and quantify multiple mRNA targets in a single RNA sample .It has high sensitivity	RNase protection assay: Does not provide information on transcript size[52]
	Real-time RT-PCR: cDNA are synthesized by reverse transcription from the sample RNA identified. The resulting cDNA is amplified by using fluorescently labeled oligonucleotide primers. Fluorescence intensity is monitored and correlated with several PCR cycles	Real-time RT-PCR: Allows quantitative genotyping, detection of SNPs and allelic variants or genetic variations even when mutation is found in very small fraction of cells in the sample. Has become clinical standard for diagnoses in Infectious diseases and it’s role is evolving rapidly in cancer diagnostics[50]	Real-time RT-PCR: The process is complex and any errors in choice of reagents, primers or probes will affect accuracy. There could be risk for errors during data analysis and reporting. The process is expensive[53]
	In situ hybridization: Tissue specimen is fixed to preserve morphology and then treated with proteases. A labeled probe is hybridized to the sample and detected using chemiluminescence or autoradiography[48]	In situ hybridization: Very useful in diagnostic application when there is limited tissue sample (in embryos and biopsy specimen). Several specific hybridizations can be done on the same sample. Tissue samples can be freeze for future use[48]	In situ hybridization: Low diagnostic yield when the sample has low DNA and RNA copies[48]
	Spotted DNA arrays: Measures relative expression levels between 2 samples. cDNA probes amplified by PCR are spotted on a glass slide and then mRNAs are isolated from the samples. The mRNA from each sample is labeled with different fluorescent dyes. The samples are mixed, co-hybridized with cDNA probes on glass slides to measure relative gene expression	Spotted DNA arrays: The major application of DNA array is measurement of gene expression levels[51]	Spotted DNA arrays: DNA array can only detect known sequences, that were used to construct the array. It only gives relative estimate of gene expression and not reliable for absolute quantification. When the genome has multiple related sequences then design of array that distinguishes these sequences is challenging. Difficult to reproduce the array[51]

SNP: Single nucleotide polymorphism; NGS: Next-generation sequencing; PCR: Polymerase chain reaction; RT-PCR: Real-time reverse transcription polymerase chain reaction; ssDNA: Single stranded DNA.