Table 1 Characteristics of diverse sequencing methods

Microarrays

Tiling arrays

A method based on probes for discovering transcripts from specific genomic regions.

This approach can provide in-depth analysis of transcripts from target regions of genome.

Suffer from potential noise as a result of weak binding or cross-hybridization of transcripts to probes.

[56]

Microarrays

A method based on a large number of oligonucleotide probes for performing quick global or parallel expression analysis of transcriptome.

Small size and high-throughput capabilities.

This method is not able to discover novel transcripts.

[57]

RNA-seq

RNA-seq

A technique that is currently the most widespread sequencing technology for both detecting RNA expression and discovering novel RNAs.

The method provides a global high-throughput detection amd identification of RNAs greater than 200 nt.

Its standard procedure is not suitable for detection of RNAs less than 200 nt. It also suffer from sequence errors at the reverse-transcription step or primer bias.

[58]

RNA capture sequencing

A derivative technology combining RNA-seq with tilling arrays.

The method can specifically elevate the sequencing depth of target regions.

Suffer from disadvantages of both tiling arrays and RNA-seq.

[59]

scRNA-seq

Smart-seq

A scRNA-seq method based on a full-length cDNA amplification strategy.

Provide a full-length cDNA amplification of polyadenylated RNAs.

The limitations are lack of strand-specific identification, inability to read transcripts longer than 4 kb and only for polyadenylated RNAs.

[60]

DP-seq

A scRNA-seq method using heptamer primers.

Suitable for smaller size samples or transcripts longer than 4 kb. this approach also suppresses highly expressed rRNAs in the cDNA library.

Captured RNAs are limited to polyadenylated RNAs.

[61]

Quartz-seq

A scRNA-seq method which reduces back ground noise.

Reduce background noise by using specially suppression PCR primers to reduce side products.

The method is limited to detecting polyadenylated RNAs.

[62]

SUPeR-seq

A single-cell universal polyadenylated tail-independent RNA sequencing.

Detect polyadenylated and nonpolyadenylated RNAs. Minimal rRNAs contamination.

Relatively low sensitivity for nonpolyadenylated RNAs.

[63]

RamDA-seq

A full-length total RNA-sequencing method for analyzing single cells.

High sensitivity for nonpolyadenylated RNAs. It can also uncover the dynamics of recursive splicing.

Unknown

[64]

Small RNA-seq

Small RNA-seq

A type of RNA-seq that discriminate small RNA from larger RNA to better evaluate and discover novel small RNAs.

Specifically detect and discover small or intermediate-sized RNAs with target sizes.

Adapter ligation bias lead to reverse transcription bias or amplification bias.

[65]

Single-cell small-RNA sequencing

Small-seq

A method which detect small RNAs in a single cell.

The method can detect small RNAs in a single cell.

The limination may be similar to small RNA-seq.

[66]

Nascent RNA-seq

GRO-seq

A method labeling nascent RNAs with 5Br-UTP and immunoprecipitating RNAs for sequencing.

Detect nascent RNAs and provide a genome-wide view of the location, orientation, and density of Pol II-engaged transcripts.

The method is confounded by contamination due to nonspecific binding, which could possibly result in experimental bias.

[67]

SLAM-seq

A method distinguishing nascent RNA from total RNA via s⁴U-to-C conversion induced by nucleophilic substitution chemistry.

It is an enrichment-free method which can avoid contamination induced by affinity purification.

The oxidation condition caused certain oxidative damage to guanine, which may impact the accurancy of sequencing.

[68]

TimeLapse-seq

A method distinguishing nascent RNA from total RNA via s⁴U-to-C conversion induced by an oxidative nucleophilic aromatic substitution reaction.

It is an enrichment-free method which can avoid contamination induced by affinity purification.

The oxidation condition caused certain oxidative damage to guanine, which may impact the accurancy of sequencing.

[69]

AMUC-seq

A method distinguishing nascent RNA from total RNA via transforming s⁴U into a cytidine derivative using acrylonitrile.

More efficient and reliable because it has a minimal influence on the base-pairing manner of other nucleosides.

Unknown

[70]

Identification of RNA-chromatin interaction

GRID-seq

A method that aims to comprehensively detect and determine the localization of all potential chromatin-interacting RNAs.

Use a bivalent linker to ligate RNA to DNA in situ and provide exact profiles of RNA-chromatin interactome.

Usable sequence length for mapping RNA is 18–23 bp. However, short sequence length can result in ambiguity in mapping.

[71]

iMARGI

A method providing a in situ mapping of RNA-genome interactome.

iMARGI needs less number of input cells and is suitable for paired-end sequencing.

Unknown

[72]

ChAR-seq

A chromatin-associated RNA sequencing that maps genome-wide RNA-to-DNA contacts.

Uncover chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs.

The method needs more than 100 million input cells.

[73]

Identification of RNA-RNA interaction

CLASH

A relatively early method that uses UV cross-linking to capture direct RNA-RNA hybridization.

Avoid noise from protein intermediate-mediated interactions.

This method only detects the RNA-RNA interactions base on proteins.

[74]

RIPPLiT

A transcriptome-wide method for probing the 3D conformations of RNAs stably associated with defined proteins.

The method can capture 3D RNP structural information independent of base pairing.

This method only detects the RNA-RNA interactions base on proteins.

[75]

MARIO

A method identifying RNA-RNA interactions in the vicinity of all RNA-binding proteins using a biotin-linked reagent.

This method can identify RNA-RNA interactions in the vicinity of all RNA-binding proteins.

The method only detects the RNA-RNA interactions base on proteins.

[76]

PARIS

Psoralen analysis of RNA interactions and structures with high throughput and resolution.

Directly measure RNA-RNA interactions independent of proteins in living cells.

Unknown

[77]

LIGR-seq

A method for the global-scale mapping RNA-RNA interactions in vivo.

Provide global-scale mapping RNA-RNA interactions independent of proteins in vivo

Unknown

[78]

SPLASH

A method providing pairwise RNA-RNA partnering information genome-wide.

Map pairwise RNA interactions in vivo with high sensitivity and specificity, genome-wide.

Unknown

[79]

RIC-seq

RNA in situ conformation sequencing technology for the global mapping of intra- and intermolecular RNA-RNA interactions.

The method performs RNA proximity ligation in situ and can facilitate the generation of 3D RNA interaction maps.

Unknown

[80]

RNA proximity sequencing

A method based on massive-throughput RNA barcoding of particles in water-in-oil emulsion droplets.

This method can detect multiple RNAs in proximity to each other without ligation and is fit for studying the spatial organization of RNAs in the nucleus.

Unknown

[81]

RNAs in protein complexes or subcellular structures

FISSEQ

A method that offers in situ information of RNAs at high-throughput levels.

Provide information of RNAs at high-throughput levels. Visualization.

Unknown

[82]

CeFra-seq

A method that physically isolates subcellular compartments and identifies their RNAs.

The methods have high sensitivity for low-abundance transcripts.

The method is limited to isolation protocols and the purity of resulting isolates.

[83]

APEX-RIP

A method can map organelle-associated RNAs in living cells via proximity biotinylation combined with protein-RNA crosslinking.

The technique can offer high specificity and sensitivity in targeting the transcriptome of membrane-bound organelles.

Unknown

[84]