Editing Nanopore RNA Sequencing Protocol (section)

=Troubleshooting=

Below is some general information that is helpful during the sequencing run. 

* '''Temperature of the MinION should be stable (a horizontal line)'''. If the temperature of your ''MinION'' is fluctuating during the sequencing run, that indicates that your ''MinION'' is having difficulty keeping a consistent temperature and may be defective. Check to ensure that the fans on the ''MinION'' itself are coming on, otherwise contact ''ONT'' for support and/or a replacement.
* '''Large amount of pores in the Inactive state'''. It has been advised from David Eccles (see this [https://community.nanoporetech.com/posts/large-proportion-of-inacti post here]) that a large amount of inactive pores in random spots indicates a defective flow cell ([https://ont-experimentcompanion-live.s3.amazonaws.com/2022/05/31/22/40/46/04b390f2-6e58-48de-8757-90fabf0816fd/1654036845853.png example here]). Issues with sample preparation and the introduction of air bubbles can cause pores to be inactive, however, this is more likely to occur in distinct regions of the flow cell. ''It is advised that you start a sequencing run without adding any flush buffer or sample to see how many pores are "available" at the start of your run'', then load as necessary. You ''should'' see a sea of green pores at the start with no issues ([https://images.ctfassets.net/76r1b51it64n/2aAOuVa2KspBdt75eNdepi/bc374ea3f73da7977e3669465b30e08e/Channel_states_panel.png example here]).
* '''Sequencing RNA will always have a decent number of pores sequencing adapter'''. Per ''ONT'' [https://community.nanoporetech.com/docs/prepare/library_prep_protocols/experiment-companion-minknow/v/mke_1013_v1_revcv_11apr2016/troubleshooting-your-run-from-the-duty-time-plots here]: "For RNA in particular, the expanded channels view may show a large proportion of pores sequencing Adapter. This happens because RNA strands are usually shorter than DNA, and the adapter takes up a larger proportion of the strand. Additionally, the RNA sequencing chemistry is optimised for sequencing RNA, whereas the adapter is DNA, and is processed slower. As long as the 'basic' pore activity plot view shows the majority of pores in "Sequencing", a high proportion of Adapter should not be a problem."
* '''According to Kanhav Khanna, an ONT tech, total RNA and mRNA perform the same for human but not for other species.''' In my discussions with him, some individuals have reported better results with mRNA (for instance, when studying RNA from yeast), but this hasn't been the case with human RNA. This appears to reflect my runs thus far.
* '''For the Flongle, aim to load 3-20 fmol of RNA; 10-15 fmol ideally.''' This can be calculated using the [https://nebiocalculator.neb.com/#!/ssrnaamt NEBio RNA mass calculator], or the formula on that page. Typically, the average RNA length (N50) is around 1,400 bases, so substitute that into the formula and 10 fmol to determine what amount you should load in ng. 
* '''"Wildflower" Pattern and Flow Cell Quality.''' Some flow cells appear to have a "wildflower pattern" as it is known on the ''ONT community forums'', since the flow cell has many pores in different states (Active Feedback, Inactive, Sequencing, Adapter, Zero, etc.). You may find a discussion about this on the [https://community.nanoporetech.com/posts/wildflower-flow-cell-patte forums here]. As noted by a user, "The good flow cells seem to have a high number of pores at initial check (~1600) whereas flow cells just above the warranty tend to fall off quickly. I saw it with flongles too, those with >80  pores were great, those starting around ~50 died quickly. It seems to be a quality control thing." You should start "sequencing" on your flow cell ''always'' to truly check the flow cell's status prior to sample loading.
* '''Quick Pore Decay can be due to a Number of Factors.''' Some of these include: large amounts of fragmented RNA/DNA in the sample (you can check this by mapping the control RCS RNA in the Direct RNA Sequencing kit and looking for fragmentation), contaminants present in your sample (detergents, proteins, etc.), poor flow cell quality (flow cells that are old and have been sitting for more than a month, or have low pore availability even if above warranty; see "wildflower" comment above), overloading the flow cell, and/or damaging the pores upon sample loading.

{| class="wikitable"
|+Nanopore Pore Statuses
|-
! scope="col"| Pore Status
! scope="col"| Description
|-
|'''Sequencing'''
|Indicates that the pore is currently sequencing. This is the ideal state. Can be either "adapter" sequencing or "strand" sequencing. You can figure out which it is by clicking the "show detailed" box.
|-
|'''Pore Available'''
|This means that the pore is available for sequencing, but no strand is currently being sequenced. '''A large portion of pores in this state usually indicates that you have loaded a small amount of library'''.
|-
|'''Adapter'''
|The pore is sequencing the unligated sequencing adapter only. Reads will initially be classified as adapter until the DNA/RNA strand starts translocating through the pore and MinKNOW is able to reclassify the read. A large portion of adapters sequencing relative to actual sample RNA means that your library preparation was pore.
|-
|'''No Pore'''
|No pore was detected in the channel.
|-
|'''Unavailable'''
|The channel appears to show a pore that is currently unavailable for sequencing.
|-
|'''Unclassified'''
|Pore status unknown.
|-
|'''Saturated'''
|The channel has switched off due to current levels exceeding hardware limitations.
|-
|'''Out of range-high'''
|Current is positive (between 10 and 9999 pA) and unavailable for sequencing.
|-
|'''Out of range-low'''
|Current is negative (between -5 and -9999 pA) and unavailable for sequencing.
|-
|'''Zero'''
|Pore currently unavailable for sequencing and the current level is between -5 and 10 pA. According to ''ONT'' specialist Kanhav Khanna, this is usually due to osmotic imbalance caused by an air bubble or some detergent in the sample. Look for "swaths" or patches of pores in the Zero state, which may indicate an air bubble in that location.
|-
|'''Multiple'''
|Multiple pores detected. Unavailable for sequencing.
|-
|'''Active Feedback'''
|The channel is reversing the current flow to eject the analyte (DNA/RNA).
|}