Advances on the MIC's n6mA
Hemi-methylated AT sites in the MAC

PhD Project

Biggest question: How does the cell recognize the Scan-RNA independant IES ?

2 independant hypothesis:

Some motifs or specific nucleotide composition

Permanently epigenetic marking - DNA methylation

PacBio SMRT sequencing

- Inter-Pulse Duration (IPD) ⇈ if n6mA

- Compared with Control or in-sillico control --> IpdRatio

- Each molecule --> analyzed independantly

- Each nucleotide --> Analyzed independantly

- Each strand --> analyzed independantly (then paired)

> 99% accuracy

~15% error

Strategy:

1:200

Random sampling

PacBio SMRT

1 out of 200 comes from the MIC
1/6 of MIC inserts will carry an IES
1/2 of IESs are just wrongly excised
30% of the remnants are scanRNA dependant

Only a few remaining: ~ 10 to ~100 sequences

If 100% carry a methylation pattern, this is enough

Sorting

Deduced origin

MIC DNA

Alignment of consensus

MAC, MIC,

Mito...

Analysis

Report n6mA

5mC, 4mC

Re-alignment

Available data at day0

Wild type:

Vegetative Cell (HTVEG)
During autogamy (after 2h - HT2)
During autogamy (after 6h - HT6)
- Asynchronous cultures / Quite blurred state

Silencing of methylase candidates:

Si/MAB (identified since then - probably a histone methylase)
MT proteins:
- Si/MT2
- Si/MT1A-1B
- Si/MT1A-1B-2
NM proteins:
- Si/NM4-9-10
- Si/NM9-10
- Si/NM4

Determining Se and Sp

Sensitivity, Specificity of n6mA detection ?
- No data for our approach in the litterature
  - Short inserts
  - Sequel vI.0
- Can't afford a real benchmarking

Paramecium is fed with E.coli !
- ~ 100% n6mA in GATC
- EcoK1 methylation well documented
- Benchmark on E.coli ?

Found:

Se ~ 93% = P(D+ | M)
Sp ~ 99.9% = P(D- | NM)

Detected level VS real level

$$p= [ Se \cdot \pi + (1 - Sp) \ (1 - \pi) ] \cdot N$$

True positives

False positives

p : Number of D+

N: Number tested

$$\pi = \frac{\frac{p}{N} - 1 + Sp}{Se-1+Sp}$$

--> From imperfect detections, allows to estimate the fraction of nucleotides truly methylated

$$\pi$$ is the true proportion of n6mA

Quantitative estimation of the n6mA in the MAC

Mostly in AT sites (~90%)

"Lots" are symmetrical (~80%)

Quantitative estimation of the n6mA in the MIC

Quantitative estimation of n6mA in the MIC (details)

Distribution of the detections

HTVEG

MT2

etc...

--> Some molecules carry all the detections, in sym-A*T

Very likely to be sequences comming from the MAC

What else could we explore ?

In the MIC:

Other type of DNA methylation
Kinetic signal around the IES
That's it

What else could we explore ?

In the MAC: Hemimethylation

Thanks !

Output example

DNA n6mA

Analysis of Paramecium tetraurelia

Enfin !

Per experiment comparaison (1)

Per-experiment comparaison (2)

In the vegetative MAC

~95% of the methylation locates in AT dinucleotides in the MAC(*)
- slightly lower in the MIC (5 to 5 points less)
- True in any condition
75% of the methylation in an AT dinucleotide is actually symetrically modified, independantly from being in the MAC or the MIC(*)