Nov 13, 2022
Version 1
Downstream Analysis of ebFRET Data V.1
This protocol is a draft, published without a DOI.
- 1University of Pennsylvania
Protocol Citation: Clark Fritsch 2022. Downstream Analysis of ebFRET Data. protocols.io https://protocols.io/view/downstream-analysis-of-ebfret-data-ci8juhun
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 13, 2022
Last Modified: November 13, 2022
Protocol Integer ID: 72683
Abstract
This protocol follows from the "Using ebFRET for Hidden Markov Modeling" protocol and is the fourth and final step towards analyzing your single-molecule FRET traces using Hidden Markov Modeling. In this protocol, I describe some of the questions that you may ask of the data generated by ebFRET and list some of the programs that I have made to do the calculations required to answer these questions.
After creating your "2stateHMM_practice_Analysis.dat", as described in the "Using ebFRET for Hidden Markov Modeling" protocol, you can then calculate / retrieve various types of information from your data.
The downstream analysis that I have done so far for the ebFRET data can be divided into the following questions:
1. What is the distribution of FRET values for each state that was identified by ebFRET?
ebFRET_HMM_State_Histograms.R 2. What is the idealized FRET efficiency for each FRET state that was identified by ebFRET?
FRET_Determination_ebFRET_v1.R 3. What is the actual FRET efficiency (non-idealized FRET) for each FRET state that was identified by ebFRET?
FRET_Determination_ebFRET(Actual FRET, Not Viterbi Mean).R 4. What is the lifetime of each FRET state that was identified by ebFRET? Outputs a file that can be used in MEMLET to determine rate constants for each state.
Rate_Determination_ebFRET_v2.R Each of the programs included above were made for simple ad-hoc analysis of the data outputted from ebFRET and will likely need to be modified slightly depending on the number of states that you had ebFRET fit to your data.