Align mbdbs data to publicly available mito genome
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To estimate conversion efficiency
@sr320 I presume I can simply use the file Mmag_mito.fa you added to our repo for this task?
Yes
Aligned the MBDBS data to the mitochondrial genome using the script bismark_mitochondria.sh. Bismark reports for each sample and the summary report and a MultiQC report are all located in results/bismark/mito. Mitochondria % CpG methylation isn't zero, but ranges from 2.8% - 27.5% and is on average 13.9%. Mitochondrial CpG methylation percentage correlates closely with %CpG methylation across the whole genome, see figure ("included" in the figure legend means "included in analysis"):
Thanks for doing this, Laura! Wow, that is def. not "zero methylation". What's really crazy for these is how high the CHG and CHH methylation is, I think CHG <2% for the nuclear genome and >20% for mito. ..more ques than answers :)
I think we need a table of CG CHG CHH methylation context for all samples, for both full genome and mitochondria to confirm sample integrity.
| Prep Sample # | Collection Sample # | pCO2 | Rep | tank | Stage | DNA batch | % Alignment (genome) | % CpG meth (genome) | % CHG meth (genome) | % CHH meth (genome) | % alignment (mito) | % CpG meth (mito) | % CHG meth (mito) | % CHH meth (mito) | included in analysis? |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | CH01-06 | Low | LC | 3 | J7 | 1a | 31.1 | 73.4 | 1.3 | 7 | 0.11 | 20.6 | 26.5 | 47.8 | yes |
| 2 | CH01-14 | Low | LC | 3 | J6 | 3a | 26.5 | 67.7 | 1.7 | 12.8 | 0.13 | 20.6 | 25.7 | 46.8 | yes |
| 3 | CH01-22 | Low | LB | 1 | J6 | 2b | 30 | 66.2 | 1.2 | 8.2 | 0.12 | 14.6 | 19.9 | 45.3 | yes |
| 4 | CH01-38 | Low | LB | 1 | J6 | 1b | 30.4 | 68.1 | 1.6 | 12.2 | 0.13 | 24.8 | 31.2 | 48.3 | yes |
| 7 | CH03-33 | Low | LB | 1 | J6 | 3b | 30.8 | 72.5 | 1.1 | 5.6 | 0.09 | 11.6 | 16.8 | 44.7 | yes |
| 12 | CH07-06 | Low | LC | 3 | J7 | 2a | 30.7 | 64.1 | 1.3 | 9 | 0.13 | 13 | 19.7 | 45.4 | yes |
| 10 | CH05-21 | High | HB | 2 | J7 | 3a | 31.1 | 64.1 | 1.3 | 12.5 | 0.12 | 12.2 | 18.7 | 47.1 | yes |
| 11 | CH05-24 | High | HC | 4 | J6 | 1a | 31.5 | 71.6 | 1.6 | 13.9 | 0.13 | 27.5 | 31.4 | 50.3 | yes |
| 15 | CH09-02 | High | HC | 4 | J6 | 3b | 29.7 | 68.4 | 1.8 | 15.6 | 0.16 | 23.1 | 26.4 | 47.6 | yes |
| 17 | CH09-28 | High | HA | 6 | J6 | 4a | 31.7 | 67.6 | 1.7 | 7.8 | 0.13 | 14.8 | 22.9 | 46.3 | yes |
| 18 | CH10-01 | High | HB | 2 | J6 | 4a | 35.3 | 70.8 | 1.4 | 7.7 | 0.16 | 19.2 | 26 | 47.6 | yes |
| 20 | CH10-11 | High | HA | 6 | J6 | 3b | 31.9 | 67.7 | 1.5 | 12.3 | 0.16 | 21.9 | 26.6 | 46.1 | yes |
| 5 | CH03-04 | Low | LB | 1 | J7 | 1b | 23.5 | 11.4 | 2.5 | 35.4 | 0.16 | 5 | 9.1 | 40.8 | no |
| 6 | CH03-15 | Low | LB | 1 | J6 | 2a | 19.1 | 19.4 | 1.5 | 18.4 | 0.13 | 4.4 | 8.7 | 37.5 | no |
| 13 | CH07-11 | Low | LC | 3 | J6 | 3a | 18.1 | 7.8 | 1.6 | 12.7 | 0.12 | 3.5 | 7 | 33.1 | no |
| 14 | CH07-24 | Low | LB | 1 | J7 | 2a | 18.1 | 7 | 1.6 | 13.7 | 0.15 | 5.1 | 9.7 | 37.4 | no |
| 8 | CH05-01 | High | HB | 2 | J7 | 1a | 28.2 | 51.8 | 2.1 | 26.6 | 0.14 | 16.2 | 25 | 48.2 | no |
| 9 | CH05-06 | High | HA | 6 | J7 | 3a | 16 | 7 | 1.4 | 16 | 0.12 | 2.8 | 5.5 | 30.9 | no |
| 16 | CH09-13 | High | HA | 6 | J7 | 2b | 12 | 45.8 | 1.9 | 19.7 | 0.05 | 10.4 | 13.6 | 41 | no |
| 19 | CH10-08 | High | HA | 6 | J6 | 2b | 27.9 | 31.2 | 1.8 | 26.8 | 0.14 | 6.7 | 13 | 43.9 | no |
Also uploaded the table to the repo here: results/alignment-meth-summary.csv
Simpler table sorted by genome CpG % methylation rate:
| Sample | pCO2 | Genome Alignment % | Genome % CpG | Genome % CHG | Genome % CHH | Mito Alignment % | Mito CpG % | Mito CHG % | Mito CHH % | included? |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | L | 31.1 | 73.4 | 1.3 | 7 | 0.11 | 20.6 | 26.5 | 47.8 | yes |
| 7 | L | 30.8 | 72.5 | 1.1 | 5.6 | 0.09 | 11.6 | 16.8 | 44.7 | yes |
| 11 | H | 31.5 | 71.6 | 1.6 | 13.9 | 0.13 | 27.5 | 31.4 | 50.3 | yes |
| 18 | H | 35.3 | 70.8 | 1.4 | 7.7 | 0.16 | 19.2 | 26 | 47.6 | yes |
| 15 | H | 29.7 | 68.4 | 1.8 | 15.6 | 0.16 | 23.1 | 26.4 | 47.6 | yes |
| 4 | L | 30.4 | 68.1 | 1.6 | 12.2 | 0.13 | 24.8 | 31.2 | 48.3 | yes |
| 20 | H | 31.9 | 67.7 | 1.5 | 12.3 | 0.16 | 21.9 | 26.6 | 46.1 | yes |
| 2 | L | 26.5 | 67.7 | 1.7 | 12.8 | 0.13 | 20.6 | 25.7 | 46.8 | yes |
| 17 | H | 31.7 | 67.6 | 1.7 | 7.8 | 0.13 | 14.8 | 22.9 | 46.3 | yes |
| 3 | L | 30 | 66.2 | 1.2 | 8.2 | 0.12 | 14.6 | 19.9 | 45.3 | yes |
| 10 | H | 31.1 | 64.1 | 1.3 | 12.5 | 0.12 | 12.2 | 18.7 | 47.1 | yes |
| 12 | L | 30.7 | 64.1 | 1.3 | 9 | 0.13 | 13 | 19.7 | 45.4 | yes |
| 8 | H | 28.2 | 51.8 | 2.1 | 26.6 | 0.14 | 16.2 | 25 | 48.2 | no |
| 16 | H | 12 | 45.8 | 1.9 | 19.7 | 0.05 | 10.4 | 13.6 | 41 | no |
| 19 | H | 27.9 | 31.2 | 1.8 | 26.8 | 0.14 | 6.7 | 13 | 43.9 | no |
| 6 | L | 19.1 | 19.4 | 1.5 | 18.4 | 0.13 | 4.4 | 8.7 | 37.5 | no |
| 5 | L | 23.5 | 11.4 | 2.5 | 35.4 | 0.16 | 5 | 9.1 | 40.8 | no |
| 13 | L | 18.1 | 7.8 | 1.6 | 12.7 | 0.12 | 3.5 | 7 | 33.1 | no |
| 14 | L | 18.1 | 7 | 1.6 | 13.7 | 0.15 | 5.1 | 9.7 | 37.4 | no |
| 9 | H | 16 | 7 | 1.4 | 16 | 0.12 | 2.8 | 5.5 | 30.9 | no |
Mitochondrial alignment is not helpful for conversion efficiency estimation, but is helpful to look at consistency across samples.