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. 2021 Sep 16;1(5):100081. doi: 10.1016/j.crmeth.2021.100081

Table 1.

Advantages and disadvantages of TSS mapping approaches

Enzymatic approach Methods General comments Method-specific features
Oligo-capping TSS-seq, PEAT, CapSeq, TL-seq, TIF-seq, Start-seq, SMORE-seq Removal of m7G cap prior to reverse transcription reduces prevalence of the 5′ G artifact, thus providing high TSS specificity. However, oligo-capping methods generally have high total RNA input requirements (see Table 2), complex protocols, and may suffer from the sequence biases of ligases used to attach oligo caps.
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    TIF-seq/SMORE-seq: simultaneous mapping of 5′ and 3′ ends of transcripts.

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    Start-seq: enhanced TSS specificity due to isolation of short transcripts from nuclear RNA.
Cap-trapping nAnT-iCAGE, SLIC-CAGE, MAPCap Oligo-capping methods generally have lower input requirements than cap-trapping methods (see Table 2) and provide high spatial resolution and sensitivity but suffer from the 5′ G artifact due to reverse transcription of capped RNA. Cap-trapping-based protocols are relatively complex and can be expensive.
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    SLIC-CAGE: uses selectively degradable carriers to facilitate processing of very small amounts of input RNA.

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    MAPCap: isolation of capped RNA with m7G immunoprecipitation versus the cap oxidation, biotinylation, and streptavidin pulldown used in CAGE methods simplifies this portion of the protocol; reduced prevalence of 5′ G artifact due to RT reaction conditions.
Template-switching reverse transcription nanoCAGE-XLa, nanoCAGE 2017a, RAMPAGEa
Tn5Prime, nanoPAREa, STRIPE-seq		 TSRT-based approaches generally have the lowest input requirements of all TSS mapping methods (SLIC-CAGE excepted, see Table 2). Their protocols tend to be simpler than those of oligo-capping and cap-trapping methods. NanoCAGE 2017, Tn5Prime, and nanoPARE use Tn5 tagmentation for library preparation, while STRIPE-seq uses stringent bead purifications to optimize library size distribution. These methods may suffer from reduced sensitivity in complex transcriptomes and are susceptible to the 5′ G artifact. In addition, several TSRT-based methods use custom sequencing primers, complicating pooling with other types of libraries.
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    nanoCAGE-XL: the companion software, CapFilter, uses the 5′ G artifact as a “cap signature” to enhance TSS detection.

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    RAMPAGE: combines cap-trapping and TSRT for enhanced TSS specificity.

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    nanoPARE: enables parallel profiling of gene body RNA signal from a single sample; companion software provided (EndGraph).

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    STRIPE-seq: very simple and rapid protocol; low cost; companion software provided (GoSTRIPEs/TSRexploreR).
a

Indicates that a custom sequencing primer is required for libraries of this type.