Cracking the Code of Human Diseases Using Next-Generation Sequencing: Applications, Challenges, and Perspectives
Table 1
Currently available NGS-based approaches for the study of the molecular basis of human diseases at DNA level.
Enrichment system
DNA input
Analytical time
Sensitivity
Specificity
Max target size
Pros/cons
Long PCR
5 ng/amplicon
4-5 h
High
High
Depends on amplicon length
Rapid analysis of large genes/excellent quality of template DNA required; longer read lengths; greater fidelity and higher yields of Taq DNA polymerase; optimization of primer design and amplification conditions.
Multiplex PCR
5 ng/multiplex
3-4 h
High
High
Depends on amplicon length and multiplexing
Simultaneous analysis of several different targets; significant time and cost reductions/primer design and amplification conditions optimization; analysis of specific genes sequences only.
Microdroplet PCR
1.5 g
48 h†
High
High
Up to 20,000 genomic loci
Analysis of a large number of genes in a single sequencing run; rapid high-quality data production/specific instrument acquisition required; high-quality DNA required; complex data analysis; high costs.
WES
500 ng–2 g
92 h
High
>60%
50–75 Mb
High coverage in targeted regions; reduced costs for large genome analysis/mutations in regulatory regions will be missed; hard to identify structural variants and copy number variations; high sequencing depth required; complex data analysis.
Targeted capture
500 ng–2 g
92 h
High
>60%
Up to 50 Mb of custom regions
High resolution; cost reduction by pooling tagged samples; less susceptible to contamination and to mismatches/large amount of high-quality DNA required; influenced by repeating elements and high values of guanine-cytosine content; complex data analysis.
WG bisulfite sequencing
50 ng–1.5 g
30 h
High
95% of CpGs
WG
High resolution and quantitative data/high DNA and sequencing coverage requirements; influenced by efficiency of bisulfite conversion; high cost per sample.
WES, whole-exome sequencing; WG, whole genome; Mb, megabase. In hours (h) and without considering sequencing time. †Calculated considering 48 samples amplified/run. Depending on the design and on the commercial enrichment technology used.
