NET-Seq

Pseudo-Seq

mNPseudo-Seq detects pseudouridylation sites in ncRNAs with single-nucleotide resolution using high-throughput sequencing. Pseudo-Seq is very similar to PSI-seq, in that both methods use CMC to modify pseudouridines selectively and halt reverse transcription. However, Pseudo-Seq circularizes cDNA strands before PCR amplification and purification, instead of using ARTseq. Briefly, poly(A)-selected RNA is fragmented and treated with CMC. The RNA is dephosphorylated with CIP and PNK, and size-selected. Next, 3' adapters are ligated to RNA strands and reverse transcription is initiated. The truncated cDNAs resulting from CMC-modified pseudouridines are purified, circularized, and PCR-amplified. The purified cDNA libraries are sequenced by an NGS method.

Similar methods: PSI-seq, Ψ-Seq, CeU-Seq.

Pros:

• Identifies pseudouridylation sites in ncRNAs
• Provides single-nucleotide resolution
• Identifies peaks by computationally calculating the ratio of reads at the initial mapped position to the total number of reads covering that position¹

Cons:

• Circularization step may introduce additional bias
• Not yet adopted widely by the scientific community

• Pseudo-Seq: Carlile T. M., Rojas-Duran M. F., Zinshteyn B., Shin H., Bartoli K. M. and Gilbert W. V. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature. 2014;
• 1. Zaringhalam M. and Papavasiliou F. N. Pseudouridylation meets next-generation sequencing. Methods. 2016;107:63-72

1. Zaringhalam M. and Papavasiliou F. N. Pseudouridylation meets next-generation sequencing. Methods. 2016;107:63-72

1. Carlile T. M., Rojas-Duran M. F., Zinshteyn B., Shin H., Bartoli K. M. and Gilbert W. V. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature. 2014;