NGS
Published:
| Layer / Goal | Widely used method(s) | Paired newer method(s) | Why the newer one is superior | Main purpose |
|---|---|---|---|---|
| DNA: genome & SVs | Short-read WGS (Illumina) | Long-read WGS (PacBio HiFi, ONT Q20+); hybrid assemblies | Resolves repeats & SVs; phasing/haplotypes; fewer mapping ambiguities | Detect SNVs, indels, SVs, CNVs, haplotypes |
| DNA: methylation | WGBS / RRBS | EM-seq / TAPS; ONT direct 5mC calling | Less DNA damage, GC bias; direct multi-base detection | Base-level 5mC/5hmC methylome and allele-specific methylation |
| Chromatin accessibility | ATAC-seq / scATAC | Multiome (ATAC+RNA); long-read ATAC | Joint regulatory info; better peak-to-gene linkage | Identify open chromatin regions, TF footprints |
| Protein–DNA binding | ChIP-seq | CUT&Tag / CUT&RUN | Higher signal-to-noise, lower input, fewer artifacts | Map TF or histone mark binding sites |
| 3D genome | Hi-C (in situ) | Micro-C; HiChIP; single-cell Hi-C | Nucleosome resolution, mark-anchored loops, single-cell structure | Reveal chromatin loops, domains, and genome folding |
| RNA: expression | RNA-seq (ribo-minus, stranded) | Long-read RNA-seq (Iso-Seq, ONT); UMI-based high-throughput | Full-length isoforms, better quant accuracy | Quantify transcript abundance and differential expression |
| RNA: isoforms & splicing | Junction-aware short-read RNA-seq | Long-read RNA/cDNA sequencing | Unambiguous isoforms, fusion/splice variant resolution | Study alternative splicing, fusions, and isoform diversity |
| Nascent transcription / kinetics | PRO-seq / GRO-seq | TT-seq; SLAM-seq / TimeLapse-seq | Time-resolved labeling; synthesis/decay rates | Measure transcriptional dynamics, initiation, and elongation |
| RNA–protein binding | eCLIP / iCLIP | iCLIP2 / irCLIP / RNP-MaP | Higher positional accuracy, captures weak interactions | Identify RNA-binding protein targets and motifs |
| RNA modifications (post-RNA) | MeRIP-seq (m6A-IP) | miCLIP2; direct RNA nanopore sequencing | Base-level m⁶A/ψ/m⁵C detection; antibody-free | Map chemical RNA modifications (m⁶A, m⁵C, ψ) |
| Translation | Ribo-seq | QTI/GTI-seq; disome-seq | Detects initiation, ribosome collisions | Measure translation efficiency, start-site mapping |
| Proteome quantification | DDA LC-MS/MS | DIA/SWATH; PASEF-DIA; single-cell proteomics | Fewer missing values, reproducible quantification | Identify and quantify proteins in bulk or single cells |
| Protein post-translational modification (PTM) | Phospho-enrichment + DDA | DIA phospho-proteomics; FAIMS/tims ion-mobility | Higher PTM coverage, multiplexing, improved quant | Identify and quantify phospho/acetyl/ubiquitin sites |
| Protein interactions / proximity | AP-MS / Co-IP-MS | BioID / TurboID / APEX | Captures transient and weak interactors; spatial context | Detect protein–protein interactions and local neighborhoods |
| Spatial multi-omics | Visium (10x) | Slide-seqV2; MERFISH / CosMx / Xenium | Higher plex & subcellular resolution; multi-modal | Map RNA/protein distribution within tissue context |
| Single-cell multi-omics | Separate scRNA/scATAC | Same-cell multiome (ATAC+RNA), SHARE-seq, Paired-Tag | Links regulatory chromatin to RNA output directly | Integrate regulatory and expression layers per cell |
┌────────────────────────────────────────────────────────┐
│ GENOME / DNA │
└────────────────────────────────────────────────────────┘
│
│ ┌──────────────────────────────────────────────────┐
▼ │ │
┌───────────────┐ ┌─────────────────────┐ ┌────────────┐
│ WGS (Illumina│ │ Methylation assays │ │ Chromatin │
│ Long-read HiFi│ │ (WGBS, EM-seq, │ │ accessibility│
│ /ONT) │ │ ONT 5mC) │ │ (ATAC-seq) │
└───────────────┘ └─────────────────────┘ └────────────┘
│ │ │
├──────────────┬────────────┴──────────┬────────┤
│ │ │
Protein–DNA binding 3D chromatin Multiome link
(ChIP-seq / CUT&Tag) (Hi-C / Micro-C) (ATAC + RNA)
│
▼
┌────────────────────────────────────────────┐
│ TRANSCRIPTOME │
└────────────────────────────────────────────┘
│
┌────────────────────┬────────────────────────────┬───────────────────┐
│ │ │ │ RNA expression & isoforms Nascent transcription RNA–protein binding RNA modifications (RNA-seq / Iso-Seq) (PRO-seq / TT-seq) (eCLIP / iCLIP2) (MeRIP / miCLIP /
nanopore direct)
│ │ │ │
└────────────────────┴─────────────┬──────────────┴───────────────────┘
│
▼
┌────────────────┐
│ TRANSLATION │
│ (Ribo-seq, │
│ disome-seq) │
└────────────────┘
│
▼
┌──────────────────────────┐
│ PROTEOME │
│ (DIA / SWATH, PTMs, │
│ single-cell MS) │
└──────────────────────────┘
│
┌─────────────────┴──────────────────┐
│ Protein interactions / proximity │
│ (AP-MS, BioID, APEX) │
└────────────────────────────────────┘
│
▼
┌───────────────────────────┐
│ Spatial multi-omics │
│ (Visium, MERFISH, CosMx) │
└───────────────────────────┘