. 2019 Oct 15;7(1):26–37. doi: 10.1016/j.gendis.2019.10.002

Table 2.

Advantages and limitations of different genomic, transcriptomic and proteomics based platforms in Common variable immunodeficiency disease. ELISA, Enzyme-linked immunosorbent assay; SNP, Single nucleotide polymorphism.

Technology	Basic technique	Advantages	Limitations	Ref
Genome-wide Association Studies	➢ Identify common genetic variants (>5% allele frequency) ➢ Variations in SNPs analyzed throughout genome ➢ Frequent genetic variations considered pointers of disease causing loci	➢ Hypothesis free approach ➢ Low cost ➢ High resolution ➢ Many loci for single trait concurrently analyzed ➢ Large number of genes can be studied concurrently	➢ Validation of results required in large data sets and different population ➢ Detects association and not causation ➢ Not predictive and explain less heritability	³²^,64, 65, 66, 67
Sanger Sequencing	➢ Fluorescent dye-labeled bases ➢ DNA fragments read through capillary electrophoresis	➢ Long reads (~750bp) ➢ High Sensitivity ➢ High accuracy ➢ Gold Standard ➢ Can be applied to large number of patients	➢ Low throughput ➢ Time consuming ➢ Detects genetic variation in known region ➢ Cannot detect translocations ➢ Cannot detect copy number changes	³⁷^,⁴⁴^,⁴⁹^,68, 69, 70
Pyro-sequencing	➢ Sequencing by synthesis ➢ Chemiluminescent based detection	➢ More sensitive than Sanger sequencing ➢ % mutated vs. wild-type DNA	➢ Short Read length ➢ Limited to known hot-spots ➢ Limited accuracy to detect homopolymer changes ➢ Limited scalability	²⁷^,³⁵^,⁵⁵^,⁶⁰
Next Generation Sequencing	➢ Involves array based massive parallel sequencing ➢ Genomic DNA is fragmented and ligated for library preparation followed by amplification and sequencing	➢ High throughput ➢ Low background noise signal ➢ High sensitivity ➢ ➢ Large dynamic range ➢ Nano-grams of starting material required	➢ Short reads (~100-500bp) ➢ Amplification bias ➢ Massive set-up and infrastructure required ➢ Limited bioinformatics
Targeted Sequencing		➢ Detects genetic variations in pre-designed gene of interest ➢ Data is easy to handle	➢ Not useful where hot-spots or gene of interest is not known	³¹^,³⁸^,³⁹^,⁴²^,⁴³
Whole Exome Sequencing		➢ Detects genetic variations in protein-coding genome (1% of total genome) ➢ Detects nucleotide variations, small insertions and deletions	➢ Does not detect genetic variations in non-protein coding genome ➢ Gene expression regulatory regions are not detected	²⁵^,³⁷^,⁴¹^,⁴⁴^,⁴⁵^,⁴⁷^,⁴⁸
Whole Genome Sequencing		➢ Detects all genetic variations (protein coding and regulatory regions) ➢ Detects all nucleotide variations and genome reorganizations (insertion, deletion, inversion, duplication or translocations)	➢ Large size of human genome sequencing expensive ➢ Large complex data is generated	³⁰^,⁴⁸
Microarray	➢ Hybridization of complementary sequences via hydrogen bonds to immobilized DNA molecule ➢ Samples are fluorescent dyes	➢ Low cost ➢ High throughput ➢ Well-defined hybridization and analysis pipelines ➢ Easy sample preparation ➢ Large number of samples per run	➢ Analysis based on pre-defined sequence ➢ Limited dynamic range ➢ Non-specific hybridization and High background ➢ Low sensitivity ➢ High variance for low expressed genes ➢ Does not identify splice variants, paralogs and novel transcripts	⁵⁰^,⁵¹
RNA-Sequencing	➢ Quantifies and sequence RNA using Next generation technology ➢ Analyze transcriptome of gene expression pattern encoded in RNA	➢ High throughput ➢ High dynamic range ➢ High sensitivity ➢ Low background noise signal ➢ No hybridization ➢ Detects alternative spliced sites, paralogous genes, SNP and non-coding RNAs identification	➢ Protocols not fully optimized ➢ High power computing facilities required ➢ High set-up and run costs ➢ Complex computational analysis ➢ Complex analysis of splice variants	⁷^,⁵²^,⁵⁶
Epigenome profiling	➢ Quantifies DNA methylation at multiple CpG sites	➢ Sodium bisulfite treatment – Gold Standard ➢ Detects gene expression in regions with high and low CpG density ➢ Low cost	➢ Not every methylated region can be captured with affinity enrichment technique ➢ Sensitive to CpG density and copy numbers ➢ Does not identify 5 mC sites ➢ No absolute quantification of methylation levels	⁵⁵^,⁵⁶^,⁶⁰
Fourier-transform infrared (FTIR) spectroscopy	➢ Monitor biochemical changes on the basis of spectral features which reflect chemical and molecular composition	➢ Rapid ➢ Inexpensive ➢ Non-invasive technique	➢ High noise ➢ Complex data ➢ High end computational methods (chemometrics) required for data analysis	⁷¹