Sensitivity analysis

This post is intended to be a plan of the steps I need to do for sensitivity analysis I need to detect experimental limitations of my study. In other words, the sensitivity analysis will help me determine what kind of motifs I will be able to detect.

My thesis study will use synthetic degenerate fragments flanked with Illumina tags, as well as sheared genomic DNA (input DNA fragments) of naturally competent species with (Campylobacter jejuni) or without self-specificity (Thermus thermophilus, Acinetobacter baylyi). Input fragments will be then recovered from the periplasm in Rec2 knockout mutants using an organic extraction technique, to then sequenced them to a high coverage (~ 1000X) and determine presence of uptake bias.

For degenerate fragments I need to:

1. I need to determine how small the uptake motifs need to be to be able to detect them.

I have already calculated frequency distribution of a dimer AA and trimer AAA motif per 30bp, 50bp and 100bp fragments.

AA 3 figuresAAA 3 figure

 

 

 

 

 

 

 

 

An important consideration that I have to take in account is what is the probability that the motif is found also in the rest of the synthetic fragments (spacers and Illumina flanking sequences).

2. Determine how strong uptake bias needs to be to be able to detect it.

2.1. Assuming that having one motif has the same probability as having more than one.

This probability distribution seems easy to calculate since a fragment without a motif would be expected to decrease while the rest of the fragments with 1 or more motifs would increase evenly regarding to the amount that 0 motifs decreased

2.2  Assuming that probability of fragments taken up increases as number of motifs increase.

I idea I have to solve point 2.2 is that first I can assume that fragments without a motif would decrease in frequency. Now the frequency that was subtracted from the fragments with 0 motifs is distributed unevenly by a certain amount. I am not sure how to calculate this, since the amount they increased has to be proportional with the total frequency of 1 (100%)

For sheared genomic fragments I need to:

1. I need to determine how small the uptake motifs need to be to be able to detect them.

1.1 how many uptake motifs (different sizes) will be expected according to distinct average size of sheared genomic fragments.

1.2 How does coverage will affect the ability to detect different uptake motifs.

For this a given genome coverage I need to calculate the coverage per base, and estimate the amount of bases with low number of reads (below a threshold, for example 10 reads).

1.3 Which will be the average number of reads per uptake motif given that the motif increment chances to be taken up by different amounts.

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