enquire about interpretation of diff_mod rate column

[g-s-2018]

Hello,

I am trying to differentiate between nucleoside analogous and natural nucleotides based on the difference in electrical charge. But I have a couple of concerns:

1. How does xpore assign the ionic current for the entire k-mer? Is it based on the position of the nucleoside? I filtered the k-mer with the expected nucleotide analogue to be in the middle (either G or A in this table). Looking at the differential modification rate, some of the values are negative. This means the modified value is less than the unmodified value. Does this mean this position has a modified nucleoside?

2. How do you calculate the signal intensity charge for each nucleoside?

3. what does the positive values indicate?

Thanks in advance,

[yuukiiwa]

Hi @g-s-2018,

How does xpore assign the ionic current for the entire k-mer? Is it based on the position of the nucleoside? I filtered the k-mer with the expected nucleotide analogue to be in the middle (either G or A in this table). Looking at the differential modification rate, some of the values are negative. This means the modified value is less than the unmodified value. Does this mean this position has a modified nucleoside?

The current values based on the output from eventalign. differential modification rate for your two samples KO_vs_RBV_24h=mod_rate_KO - mod_rate_RBV_24h

How do you calculate the signal intensity charge for each nucleoside?

I am not sure what you mean by signal intensity charge.

what does the positive values indicate?

Positive values indicate that it is differentially modified on the KO side

Thanks!

Best wishes,
Yuk Kei

[g-s-2018]

Hi Yuk Kei,

Thanks for your clarification. In the previous question about the calculation of the signal intensity charge for each nucleoside, I meant the nucleotides exert the largest impact on the ionic current when they are in the central position of the kmer. some tools using the position of the first nucleotide in the kmer to assign all the ionic current for the entire kmer. I am unsure how xpore calculates the modification rate and how it assigns positions on the RNA strand. My question arises because I am trying to detect the incorporation of nucleoside analogues into RNA. These analogues incorporate randomly throughout the RNA strand as either G , so there's no specific position to target.

To address this, I have filtered my data to include only k-mers with G in the middle position. Additionally, I'm using in vitro transcribed RNA as a control. In this case, can I interpret any position with a negative modification rate as a potential site of nucleoside analogue incorporation?

By differentially modified position, do we mean there is a difference in the signal intensity (the electrical charge for a particular kemer) between the control vs test?

Thanks very much for your help