Table 1.
Applications of next-generation DNA sequencing
| Method | Sequencing to determine: | Example reference | ‘Subway’ route as defined in Figure 3 |
|---|---|---|---|
| DNA-Seq | A genome sequence | 57 | Comparison, ‘anatomic’ (isolation by anatomic site), flow cytometery, DNA extraction, mechanical shearing, adaptor ligation, PCR and sequencing |
| Targeted DNA-Seq | A subset of a genome (for example, an exome) | 20 | Comparison, cell culture, DNA extraction, mechanical shearing, adaptor ligation, PCR, hybridization capture, PCR and sequencing |
| Methyl-Seq | Sites of DNA methylation, genome-wide | 34 | Perturbation, genetic manipulation, cell culture, DNA extraction, mechanical shearing, adaptor ligation, bisulfite conversion, PCR and sequencing |
| Targeted methyl-Seq | DNA methylation in a subset of the genome | 129 | Comparison, cell culture, DNA extraction, bisulfite conversion, molecular inversion probe capture, circularization, PCR and sequencing |
| DNase-Seq, Sono-Seq and FAIRE-Seq | Active regulatory chromatin (that is, nucleosome-depleted) | 113 | Perturbation, cell culture, nucleus extraction, DNase I digestion, DNA extraction, adaptor ligation, PCR and sequencing |
| MAINE-Seq | Histone-bound DNA (nucleosome positioning) | 130 | Comparison, cell culture, MNase I digestion, DNA extraction, adaptor ligation, PCR and sequencing |
| ChIP-Seq | Protein-DNA interactions (using chromatin immunoprecipitation) | 131 | Comparison, ‘anatomic’, cell culture, cross-linking, mechanical shearing, immunoprecipitation, DNA extraction, adaptor ligation, PCR and sequencing |
| RIP-Seq, CLIP-Seq, HITS-CLIP | Protein-RNA interactions | 46 | Variation, cross-linking, ‘anatomic’, RNase digestion, immunoprecipitation, RNA extraction, adaptor ligation, reverse transcription, PCR and sequencing |
| RNA-Seq | RNA (that is, the transcriptome) | 39 | Comparison, ‘anatomic’, RNA extraction, poly(A) selection, chemical fragmentation, reverse transcription, second-strand synthesis, adaptor ligation, PCR and sequencing |
| FRT-Seq | Amplification-free, strand-specific transcriptome sequencing | 119 | Comparison, ‘anatomic’, RNA extraction, poly(A) selection, chemical fragmentation, adaptor ligation, reverse transcription and sequencing |
| NET-Seq | Nascent transcription | 41 | Perturbation, genetic manipulation, cell culture, immunoprecipitation, RNA extraction, adaptor ligation, reverse transcription, circularization, PCR and sequencing |
| Hi-C | Three-dimensional genome structure | 71 | Comparison, cell culture, cross-linking, proximity ligation, mechanical shearing, affinity purification, adaptor ligation, PCR and sequencing |
| Chia-PET | Long-range interactions mediated by a protein | 73 | Perturbation, cell culture, cross-linking, mechanical shearing, immunoprecipitation, proximity ligation, affinity purification, adaptor ligation, PCR and sequencing |
| Ribo-Seq | Ribosome-protected mRNA fragments (that is, active translation) | 48 | Comparison, cell culture, RNase digestion, ribosome purification, RNA extraction, adaptor ligation, reverse transcription, rRNA depletion, circularization, PCR and sequencing |
| TRAP | Genetically targeted purification of polysomal mRNAs | 132 | Comparison, genetic manipulation, ‘anatomic’, cross-linking, affinity purification, RNA extraction, poly(A) selection, reverse transcription, second-strand synthesis, adaptor ligation, PCR and sequencing |
| PARS | Parallel analysis of RNA structure | 42 | Comparison, cell culture, RNA extraction, poly(A) selection, RNase digestion, chemical fragmentation, adaptor ligation, reverse transcription, PCR and sequencing |
| Synthetic saturation mutagenesis | Functional consequences of genetic variation | 93 | Variation, genetic manipulation, barcoding, RNA extraction, reverse transcription, PCR and sequencing |
| Immuno-Seq | The B-cell and T-cell repertoires | 86 | Perturbation, ‘anatomic’, DNA extraction, PCR and sequencing |
| Deep protein mutagenesis | Protein binding activity of synthetic peptide libraries or variants | 95 | Variation, genetic manipulation, phage display, in vitro competitive binding, DNA extraction, PCR and sequencing |
| PhIT-Seq | Relative fitness of cells containing disruptive insertions in diverse genes | 92 | Variation, genetic manipulation, cell culture, competitive growth, linear amplification, adaptor ligation, PCR and sequencing |
FAIRE-seq, formaldehyde-assisted isolation of regulatory elements–sequencing. MAINE-Seq, MNase-assisted isolation of nucleosomes-sequencing; RIP-Seq, RNA-binding protein immunoprecipitation-sequencing; CLIP-Seq, cross-linking immunoprecipitation-sequencing; HITS-CLIP, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation; FRT-Seq, on-flowcell reverse transcription–sequencing. NET-Seq, native elongating transcript sequencing. TRAP, translating ribosome affinity purification. PhIT-Seq, phenotypic interrogation via tag sequencing.