TABLE 1.
Summary of major differences between the most common 3′‐end sequencing approaches
| Feature | Method | |||||
|---|---|---|---|---|---|---|
| TAIL‐Seq (Chang et al., 2014) | mTAIL‐Seq (Lim et al., 2016) | PAL‐Seq (Subtelny et al., 2014) | FLAM‐Seq (Legnini et al., 2019) | PAIso‐Seq (Y. Liu, Nie, et al., 2019) | DRS (Garalde et al., 2018) | |
| RNA preparation | Ribodepletion | Not required | Not required | Poly(A) + selection | Not required | Poly(A) + selection |
| Amount of starting material | 100 μg | <1 μg | 1–50 μg | 500 ng–10 μg | ≤100 ng | 500 ng of polyadenylated RNA |
| Sequencing platform | Illumina HiSeq/MiSeq | Illumina HiSeq/MiSeq | Illumina Genome Analyzer | Pacific BioSciences | Pacific BioSciences | Oxford Nanopore Technologies |
| 3′ adapter addition | Ligation of biotinylated single‐stranded DNA oligo | Splint ligation of biotinylated hairpin DNA oligo | Splint ligation of biotinylated single‐stranded DNA oligo in the presence of splint DNA oligo | Enzymatically added G/I tail serving as anchor for oligo | 3′‐end extension of template oligo containing T stretch | Splint ligation of DNA oligo containing T stretch |
| Fragmentation | Partial digestion with RNase T1 | Partial digestion with RNase T1 | Partial digestion with RNase T1 | None | None | None |
| PCR bias | + | + | + | + | + | − |
| RT bias | + | + | + | + | + | − |
| Oligo(dT) selection bias | − | − | − | + | − | + |
| Error rate | Low | Low | Low | Low | Low | High |
| Read length | Short | Short | Short | Long | Long | Long |
| Detection of modifications in 3′‐end of poly(A) tail | + | − | − | − | − | − |
| Detection of modifications within poly(A) tail | − | − | − | + | + | − |