Editing Nanopore RNA Sequencing Protocol (section)

=Additional Information=

One last thing I wanted to include in this protocol is a list of useful information I have found regarding running mRNA samples through the ''nanopore''. This includes references to the ''ONT'' documentation (which may require a login to access the original source) as well as publications. Reference back to it as necessary.

* '''Poly(A) Tail Requirements:''' According to ''ONT'', a poly(A) tail is required for the preparation of the RNA sequencing library. Some RNA molecules, such as long-non-coding RNAs (lncRNAs), rRNAs, tRNAs, and some of your controls (i.e. ''G. Luciferase'' and ''C. Luciferase'' RNAs) will therefore not be read. It is important, then, to add a poly(A) tail to these molecules enzymatically ''in vitro'' prior to the generation of the RNA library using an ''E. coli'' poly(A) polymerase ([https://www.neb.com/products/m0276-ecoli-poly-a-polymerase?cnum=M0276L#Product%20Information NEB Cat No.: M0276L]). Details about this procedure can be found in the ONT Documentation, which also shows a few graphs suggesting optimum incubation time (0.5-1.5 min) at 37ºC ([https://community.nanoporetech.com/knowledge/know-how/polyad-npolyA-ecoli ONT Docs: RNA Polyadenylation]).
* '''Poly(A) Tail Length Bias:''' ''ONT'' has noted in their documentation that there may be a yield bias for mRNAs that have longer poly(A) tails in yeast, but it seems that this may not be the case for humans. Please consult the documentation for more ([https://community.nanoporetech.com/knowledge/know-how/enrichment-of-polyA ONT Docs: Poly(A) Enrichment]).
* '''RNA Contaminants:''' Various chemicals used in the extraction procedure, including Phenols, Ethanol, Isopropanol, EDTA, NaCl, etc. can affect your RNA. Details of these effects can be found in the ''ONT Documentation'', but the bottom line is that you should avoid contamination during library preparation at all costs ([https://community.nanoporetech.com/rna_contaminants ONT Docs: RNA Contaminants]). Additionally, contaminants in your RNA sample may also block the pores in your flow cell, leading to a lot of pores being placed in recovery (blue).
* '''RNA Read Direction 3’->5’:''' In the RNA sequencing protocol, ''ONT'' notes the following: “''Please note that, unlike DNA, RNA is translocated through the nanopore in the 3’-5’ direction. However, the basecalling algorithms automatically flip the data, and the reads are displayed 5’-3’.''” Useful to keep in mind.
* '''RNA Stability in Storage:''' ''ONT'' describes in their documentation the stability of RNA over time. If stored properly, RNA should remain relatively stable in the proper buffers at -20ºC for 200+ days ([https://community.nanoporetech.com/knowledge/know-how/rna-stability ONT Docs: RNA Stability]).
* '''RNA Stability during Sequencing:''' RNA will degrade during the sample run over time. ''Workman et al.'' found that RNA sequences degrade over the course of a 36 hour run, but this effect was minimal (~5%) in mitochondrial control RNAs over this period, suggesting that a run over the course of 24 hours should exhibit negligible degradation ([https://www.nature.com/articles/s41592-019-0617-2 Workman et al. 2019]).
* '''RNA Quality Check:''' It is advised that the RNA samples be evaluated for quality via an RNA Integrity Number (RIN) (7+/10) prior to sequencing using an [https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument/2100-bioanalyzer-instrument-228250 Agilent 2100 Bioanalyzer]. This equipment can be pretty pricey, though. Your final RNA library to be sequenced via nanopore '''should be around 20 ng/uL''' for a good output.
* '''M6ANet Requires a Minimum of 20 Reads:''' According to the author [https://github.com/GoekeLab/m6anet/discussions/32 here], m6ANet will not generate any inferences for mapped transcripts with fewer than 20 reads. This is a limitation of the software.
* '''M6ANet Mod Probability:''' According to the author [https://github.com/GoekeLab/m6anet/discussions/114 here], the m6ANet mod probability represents the probability that the modification at that site would be picked up using m6ACE.
* '''M6ANet Mod Ratio:''' Again, according to the author [https://github.com/GoekeLab/m6anet/discussions/114 here], the m6ANet mod ratio represents the ratio of m6A modifications found to be modified 'using' the m6ANet mod probability. Therefore, mod ratio and mod probability should be positively correlated.
* '''RNA Read Errors by Base:''' RNA nanopore reads have higher error rates than for DNA nanopore reads. Additionally, not all basecalling errors are the same. For instance, the nanopore system has a higher error rate confusing C-to-U (3.62%) and U-to-C (2.23%) than G-for-C (0.38%) and C-for-G (0.47%) with Albacore ([https://www.nature.com/articles/s41592-019-0617-2 Workman et al. 2019]).
* '''RNA Read Truncation:''' In the ''Workman et al.'' study, it was also found that there were a number of truncated reads in their mitochondrial poly(A) transcripts, which were used as a control (since they are abundant, vary in length from 349-2,379 nucleotides, and are a single exon). They hypothesize that some possible non-biological causes of this include: 1) the last handful of nucleotides are not read when the pore translocation enzyme detaches the strand near the end, which causes the strand to rapidly move through the pore, 2) ionic current signal artifacts due to enzyme stalls during RNA translocation, or 3) RNA strand breaks ([https://www.nature.com/articles/s41592-019-0617-2 Workman et al. 2019]).
* '''RNA m6A Controls:''' The ''Workman et al.'' study also used an m6A synthetic oligomer (FLuc) as a control for m6A detection. Very useful for you. It is described in the ''Oligomer Ligation'' section of the Methods in their paper ([https://www.nature.com/articles/s41592-019-0617-2 Workman et al. 2019]).