---
# System prepended metadata

title: 'Nanozoo Lorena: Sample analysis Mateusz'

---

# Lorena sample analysis 
# Sample info:
## What we did:
1. We used the reference genome: GCA_000013425.1_ASM1342
https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000013425.1/
2. We reanotated the genome with Bakta (Annotation program) for additional novel transcript discovery 
3. For RNA-Seq analysis and the detection of significantlly differentially expressed genes, we used [RNAflow](https://www.mdpi.com/2073-4425/11/12/1487) with the following command:

```
nextflow run hoelzer-lab/rnaflow -r 1.4.7 --reads reads.csv \
    --genome genome.csv --annotation anno.csv -profile docker,local \
    --cores 15 --memory 128 --strand 2 \
    --featurecounts_additional_params '-t CDS -g ID'
```

Here is a detailed description of what RNAflow does step by step: https://github.com/hoelzer-lab/rnaflow
(use this also for citation)

Please note in this context, that the RNA-Seq data we produced is **strand specific** which means that we know from which strand (+ or -) a transcript originated. Thus, we also performed the read counting strand-specific. 

## Results structure 
1. The results directory includes folders 01 to 07 for analysis steps, and additional Logs (information about workflow execution) and Summary (samples quality control) folders.

```
├── 01-Trimming
├── 02-rRNARemoval
├── 03-Mapping
├── 04-Counting
├── 05-CountingFilter
├── 06-Annotation
├── 07-DifferentialExpression
├── Logs
└── Summary
```
2. Folders `01-05` contain preliminary analysis (e.g., mapping, rRNA removal).
3. Folder `06` holds Bakta annotations ('annotation.CDS.gtf'); search gene abbreviations or full names in a spreadsheet to locate gene IDs.

**Note** You will see that we produced a lot of results. Below you will find an explanation how to work with the output and what you can find in all (sub)folders. Please also note, that we usually provide output tables and plots in various formats (PNG, PDF, SVG) and with different normalization methods and filterings. While this increases the number of files, it also offers you more flexibility when investigating the data and selecting content for further downstream analysis.

> To search for a specific gene of interest, open the annotation.CDS.gtf file in a spreadsheet software (e.g., Excel). Search for the gene using its abbreviation or full name. The ID (e.g., PANCAC_00010) represents the gene in the analysis and was named by the annotation software Bakta.

4. Folder `07-DifferentialExpression` organizes differential expression by group comparison, with subfolders detailing the respective pairwise analysis (e.g., '3h_vs_6h'). Example content:
```
├── 3h_vs_6h
│  ├── input
│  │  ├── input.csv
│  │  └── input.xlsx
│  ├── plots
│  │  ├── heatmaps
│  │  ├── MA
│  │  ├── PCA
│  │  ├── sample2sample
│  │  └── volcano
│  ├── reports
│  │  ├── csslib
│  │  ├── DESeq2_results_exploration.bib
│  │  ├── DESeq2_results_exploration.html
│  │  ├── DESeq2_results_exploration.pdf
│  │  ├── DESeq2_results_exploration.Rmd
│  │  ├── figuresRNAseq_analysis_with_DESeq2_full
│  │  ├── figuresRNAseq_analysis_with_DESeq2_p0.01
│  │  ├── figuresRNAseq_analysis_with_DESeq2_p0.05
│  │  ├── jslib
│  │  ├── RNAseq_analysis_with_DESeq2_full.html
│  │  ├── RNAseq_analysis_with_DESeq2_p0.01.html
│  │  └── RNAseq_analysis_with_DESeq2_p0.05.html
│  └── results
│     ├── deseq2_3h_vs_6h_filtered_NA.csv
│     ├── deseq2_3h_vs_6h_filtered_NA.xlsx
│     ├── deseq2_3h_vs_6h_filtered_NA_extended.csv
│     ├── deseq2_3h_vs_6h_filtered_NA_extended.xlsx
│     ├── deseq2_3h_vs_6h_filtered_padj_0.01.csv
│     ├── deseq2_3h_vs_6h_filtered_padj_0.01.xlsx
│     ├── deseq2_3h_vs_6h_filtered_padj_0.01_extended.csv
│     ├── deseq2_3h_vs_6h_filtered_padj_0.01_extended.xlsx
│     ├── deseq2_3h_vs_6h_filtered_padj_0.05.csv
│     ├── deseq2_3h_vs_6h_filtered_padj_0.05.xlsx
│     ├── deseq2_3h_vs_6h_filtered_padj_0.05_extended.csv
│     ├── deseq2_3h_vs_6h_filtered_padj_0.05_extended.xlsx
│     ├── deseq2_3h_vs_6h_full.csv
│     ├── deseq2_3h_vs_6h_full.xlsx
│     ├── deseq2_3h_vs_6h_full_extended.csv
│     ├── deseq2_3h_vs_6h_full_extended.xlsx
│     └── summary.txt
├── data
│  ├── counts
│  └── input
└── plots
   ├── heatmaps
   └── PCA

```

5. The subfolder named `data` contains raw data ued for all downstream analysis (normalized read counts, ...), while the `plots` folder includes general analysis visualizations such as PCA and heatmap plots, aimed at visually demonstrating the grouping of the samples.
6. The **main results probably most interesting** for you are located within the specific group folders (e.g., `3h_vs_6h`). The main overview of the analysis is presented in the `DESeq2_results_exploration.{pdf,html}` files within the `reports` folder, where all the basic analyses are visualized and explained.
7. Additional plots (heatmaps, MA, volcano, PCA, and sample-to-sample) for more indepth gene expression analysis are found in the `plots` folder for each pairwise comparison.
8. To search for a specific gene, the Bakta ID needs to be find as detailed in point 3, then look it up in the interactive HTML file named `RNAseq_analysis_with_DESeq2_full.html` within the `reports` folder. The plot miniature near the gene is clickable for a better view of the normalized expression values. If the plot needs to be extracted, it is located in the `figuresRNAseq_analysis_with_DESeq2_full` folder.
9. The raw results displayed in the HTML file can be accessed in the `results` folder, within the file `deseq2_3h_vs_6h_full_extended.xlsx`, which includes a complete gene description (with the abbreviation and full name) in the last column. For convenience, the files named `_filtered_padj_0.XX_extended.csv` offer a prefiltered list of genes based on adjusted p-value cutoffs (0.05 which is a commonly accepted default and more restrictive 0.01).
10. Genes are considered significantly differentially expressed if the adjusted p-value (with Benjamini-Hochberg correction) (padj) is below 0.05, and the log2 fold change (log2FoldChange) is above 1 (indicating upregulation) or below -1 (indicating downregulation) of a gene.

## Interpretation

# Quality control
The quality of the run can be checked in the `Summary` folder file `multiqc_report.html`

- We observed a large quantity of % rRNA, suggesting an issue with the depletion process. However, after using SortMeRNA, the RNA samples were computationally cleaned, and no rRNA artifacts were observed in the DESeq2 analysis (Section General Statistics).
- The rRNA issue could also explain the high level of duplication observed in the FastQC analysis of trimmed sequences.
- Due to the high level of rRNA, the final sequencing depth was more shallow than expected. Nonetheless, we retained approximately 94% of the features (CDS) based on TPM (transcripts per million, a normalization meassure) filtering, indicating that sufficient reads remained for accurate expression profiling even after rRNA removal. We also expected the mapping results and saw good expression values for genes. 
- Despite the potential issues mentioned above, following the DESeq2 analysis, we detected a large portion of unfiltered features without an overestimation of small features or any single type. Most importantly, we observed good group separation (PCA plots).
- In summary, the data is of good quality and can be used for differential gene expression analysis

# Grouping
- we checked three different groups: granular comparison (every type as a separate group), time (3 vs 6h) and conditions (MIN vs CSS).

The general plots grouping plots can be viewed in the respective group folder (eg. MINvsCSS), `plots`, `PCA`.

- PCA plot shows good separation in granular comparison, with grouping over time and with same samples staying on the same plane of prinicipal component 2.
![PCA_simple_vsd_top500](https://hackmd.io/_uploads/HyFJPJvCp.svg)


- The main separation is visible towards time (40 % variance from the first component) however that could be explained purely by difference in growth phase.
![PCA_simple_vsd_top500](https://hackmd.io/_uploads/Sy3ZPyPA6.svg)


- Considering conditions (MIN vs CSS) the variability between conditions is much smaller but still observable, a lot of possible differences could be masked by time or strain effect.
![PCA_simple_vsd_top500](https://hackmd.io/_uploads/HkPMPywR6.svg)


# Gene counts

- Across all analyzed scenarios, we consistently identify a substantial number of genes that are significantly differentially expressed.

|Condition     |BH adjusted p-values < 0.05|
|-----|-----|
|MIN vs CSS    | 699    |
|3h vs 6h     | 1499    |

# CSS vs MIN

## Glycolysis and virulence

- Glycolysis and virulence factors were selected by querying the Uniprot database with the matched reference genome and matched to our annotation. 


### Glycolysis

- When examining glycolysis, no genes exhibited significant differential expression (log2FoldChange above 1 or below -1).

### Virulence
- We identified 8 features with a log2FoldChange greater than 1 (upregulation in MIN condition).

|padj|log2FoldChange|fullDescription|gene|
|-|-|-|-|
| 1.710940e-08|1.36952626707895|ID PANCAC_01150;Name WXG100 family type VII secretion effector EsxA|esxA|
|1.74817624318794e-05|2.28996883496689|ID PANCAC_12925;Name HTH-type transcriptional regulator SarU;|sarU|
|0.000829615840181056|1.07920409862855|ID PANCAC_06300;Name Nickel import system ATP-binding protein NikD|nikD|
|0.00252697794081369|1.05649660650798|ID PANCAC_01175;Name type VII secretion system protein EssC%2C FtsK/SpoIIIE family ATPase |essC|
|0.00281546417047228|1.14916708090827|ID PANCAC_12525;Name bi-component gamma-hemolysin HlgCB subunit C; |hlgC|
|0.0206923007164747|1.08002947966662|ID PANCAC_11885;Name Staphylococcal secretory antigen ssaA2;|ssaA2|
|0.0217808234988874|1.44054460864811|IID PANCAC_10030;Name chemotaxis-inhibiting protein CHIPS;|chp|
|0.0318276820094005|1.11355627226274|ID PANCAC_12515;Name immunoglobulin-binding protein Sbi;|sbi|


- We identified 7 features with a log2FoldChange lower than -1 (downregulation in MIN conditions).

|padj|log2FoldChange|Description|gene|
|-|-|-|-|
|3.209189e-09 |-4.082931|ID PANCAC_10460;Name accessory gene regulator AgrB|agrB|
|2.082704e-04|-1.73426|ID PANCAC_01730;Name superantigen-like protein SSL1|ssl1|
|1.021241e-03|-1.159935|ID PANCAC_02320;Name ATP-dependent Clp protease ATP-binding subunit ClpC|clpC|
|4.684083e-03|-1.497872|ID PANCAC_02315;Name protein arginine kinase;|mcsB|
| 1.437867e-02 |-1.204543|ID PANCAC_00325;Name staphyloferrin B biosynthesis protein SbnC;|sbnC|
|1.481039e-02|-1.172238|ID PANCAC_03725;Name MSCRAMM family adhesin clumping factor ClfA|clfA|
|2.027476e-02|-1.170879|D PANCAC_00320;Name N-[(2S)-2-amino-2-carboxyethyl]-L-glutamate dehydrogenase SbnB|sbnB|

## Top 20 most differently expressed genes 
### Upregulated (in MIN conditions)

|padj|log2FoldChange|Description|gene|
|-|-|-|-|
|3.45917651287683e-08|13.1707123454778|ID PANCAC_09345;Name putative autolysin LytO|lytO|
|9.13175589416158e-08|12.5312109278829|ID PANCAC_00540;Name Glycosyltransferase involved in cell wall bisynthesis |rfaB|
|1.41577309536786e-07|12.318122627627|ID PANCAC_00545;Name O-antigen ligase  rfaL|
|9.06535065510678e-07|11.4484484117512|ID PANCAC_00535;Name Acetyltransferase%2C isoleucine patch superfamily |wbbJ|
|3.04472864975688e-09|11.2539283465258|ID PANCAC_00135;Name TIGR04141 family sporadically distributed protein||
|1.69834341164519e-07|9.98150197406611|ID PANCAC_06915;Name Peptidase||
|3.8161558494272e-07|9.55798379998377|ID PANCAC_06885;Name DUF2951 domain-containing protein||
|3.67345364779838e-05|9.18990360914133|ID PANCAC_06940;Name Ig domain-containing protein||
|6.00170303600503e-47|9.1841024863071|ID PANCAC_02700;Name Tandem five-TM protein||
|7.85095636595535e-07|9.17230738529227|ID PANCAC_06900;Name BppU N-terminal domain-containing protein |bppU
|6.70227577399866e-07|9.13693655819812|ID PANCAC_07110;Name DNA-directed DNA polymerase||
|5.95885134201965e-05|9.11759916548978|ID PANCAC_06945;Name Major tail protein||
|3.27264438755189e-05|9.03652915306223|ID PANCAC_06950;Name DUF3168 domain-containing protein||
|0.000132172281766788|8.7178874792676|ID PANCAC_06905;Name Minor structural protein;locus_tag PANCAC_06905;product Minor structural protein||
|5.78394703006941e-06|8.71308877858962|ID PANCAC_07005;Name DNA helicase||
|0.000161085585699093|8.58635447751182|ID PANCAC_06895;Name DUF2977 domain-containing protein||
|6.90844161094887e-05|8.56902903865606|ID PANCAC_07150;Name Phage protein||
|4.40210324304235e-05|8.43526060133724|ID PANCAC_07025;Name Virulence-associated protein E-like domain-containing protein||
|0.000321370808422332|8.26206478634709|ID PANCAC_06925;Name lysostaphin||
|6.22224656748816e-06|8.25906718641754|ID PANCAC_06960;Name Phage protein||


### Downregulated (in MIN conditions)

padj|log2FoldChange|fullDescription|gene
|-|-|-|-|
1.61743933227544e-07|-7.36099951973174|ID PANCAC_06840;Name DUF1672 domain-containing protein||
3.92568432446332e-05|-5.55588993284035|ID PANCAC_01245;Name Cytosolic protein||
2.1357841895678e-08|-4.80750446488956|ID PANCAC_13090;Name L-amino acid N-acyltransferase MnaT |mnaT|
3.20918875929455e-09|-4.0829309494576|ID PANCAC_10460;Name accessory gene regulator AgrB |regulator AgrB|agrB|
2.08478824248883e-11|-3.62677271389645|ID PANCAC_04485;Name Holin-like toxin||
1.15992827167898e-09|-3.42299394917159|ID PANCAC_13990;Name DNA-binding transcriptional regulator%2C XRE-family HTH domain |xRE|
0.654822023489967|-3.39625570405407|ID PANCAC_00370;Name (S)-acetoin forming diacetyl reductase |butA|
6.081106425111e-07|-2.9856299331102|ID PANCAC_12500;Name Putative metal homeostasis protein||
0.00016203160668587|-2.26754385929842|ID PANCAC_05365;Name carbamoyl phosphate synthase small subunit |carA|
0.000402998817241426|-2.21112214569831|ID PANCAC_05360;Name dihydroorotase |pyrC|
8.05570450568469e-14|-2.18298437737955|ID PANCAC_00405;Name Uncharacterized protein SAOUHSC_00094||
0.000762618289728995|-2.15467569029024|ID PANCAC_05355;Name aspartate carbamoyltransferase catalytic subunit |pyrB|
0.0125937878608676|-2.13592266714364|ID PANCAC_10195;Name dUTPase||
0.0010215378795991|-2.12243931934099|ID PANCAC_03740;Name Extracellular matrix and plasma binding protein||
0.0025056659176384|-2.06471138178317|ID PANCAC_05350;Name Xanthine/uracil permease |uraA|
0.000197674893896122|-2.04581979448666|ID PANCAC_05370;Name carbamoyl-phosphate synthase large subunit;|carB|
0.00133675843441694|-2.02506435292983|ID PANCAC_00085;Name adenylosuccinate synthase |purA|
4.61865620166387e-05|-1.99762471791315|ID PANCAC_09855;Name cysteine protease staphopain A |scpA|
0.00105162368799605|-1.85729049983564|ID PANCAC_13510;Name betaine-aldehyde dehydrogenase;|betB|
0.674741861471901|-1.79124123363204|ID PANCAC_11005;Name DNA-binding ferritin-like protein (oxidative damage protectant)|dps|


## Analysis for Cluster of orthologs genes (COG)
Selected from adjusted p < 0.05
Further explanation can be found:
https://www.ncbi.nlm.nih.gov/research/cog/
Legend:

| Letter | Description                                                       |
|--------|-------------------------------------------------------------------|
| D      | Cell cycle control, cell division, chromosome partitioning        |
| M      | Cell wall/membrane/envelope biogenesis                            |
| N      | Cell motility                                                     |
| O      | Post-translational modification, protein turnover, and chaperones |
| T      | Signal transduction mechanisms                                    |
| U      | Intracellular trafficking, secretion, and vesicular transport     |
| V      | Defense mechanisms                                                |
| W      | Extracellular structures                                           |
| Y      | Nuclear structure                                                 |
| Z      | Cytoskeleton                                                      |
| A      | RNA processing and modification                                   |
| B      | Chromatin structure and dynamics                                  |
| J      | Translation, ribosomal structure and biogenesis                   |
| K      | Transcription                                                     |
| L      | Replication, recombination and repair                             |
| C      | Energy production and conversion                                  |
| E      | Amino acid transport and metabolism                               |
| F      | Nucleotide transport and metabolism                               |
| G      | Carbohydrate transport and metabolism                             |
| H      | Coenzyme transport and metabolism                                 |
| I      | Lipid transport and metabolism                                    |
| P      | Inorganic ion transport and metabolism                            |
| Q      | Secondary metabolites biosynthesis, transport, and catabolism     |
| R      | General function prediction only                                  |
| S      | Function unknown                                                  |

### Upregulated (in MIN conditions)
Total:

|              |      |
| ------------ | ---- |
| Assigned COG | 84      |
| NA           | 59 |
![cog_up](https://hackmd.io/_uploads/SkTIvWD0p.jpg)

### Downregulated (in MIN conditions)

|              |      |
| ------------ | ---- |
| Assigned COG | 45     |
| NA           | 26 |
![cog_down](https://hackmd.io/_uploads/rJywvbPR6.jpg)


# Time (3hvs6h)
## Glycolysis and virulence
### Glycolysis
- Time was the most influential factor for differently expression of glycolysis.
- We found 6 features with above 1 log2FoldChange (upregulation in 3h conditions).

| padj       | log2FoldChange | fullDescription                                                                                                   | gene |
|:---------- | -------------- | ----------------------------------------------------------------------------------------------------------------- | ---- |
|4.66358165218007e-29|2.12948286971182|ID PANCAC_08210;Name 6-phosphofructokinase |pfkA|
|3.75050593771086e-21|1.81914542553207|ID PANCAC_08205;Name pyruvate kinase |pyk|
|5.10751259133808e-19|2.49566541705408|ID PANCAC_03655;Name Phosphoglycerate kinase |pgk|
|3.03438617372401e-18|2.28367522010457|ID PANCAC_03660;Name triose-phosphate isomerase |tpiA|
|5.90152573281243e-18|2.24664715869237|ID PANCAC_03665;Name 2%2C3-bisphosphoglycerate-independent phosphoglycerate mutase |gpmI|
|2.40245568905275e-09|1.53977602864747|ID PANCAC_04130;Name glucose-6-phosphate isomerase |pgi|
|6.01380153103385e-07|1.08881298682941|ID PANCAC_03670;Name phosphopyruvate hydratase |eno|

- We found  1 gene with below -1 log2FoldChange (downregulation in 3h conditions).

| padj       | log2FoldChange | fullDescription | gene |
|:---------- | -------------- | ----------------------------------------------------------------------------------------------------------------- | ---- |
|1.04051239318994e-29|-3.13648872140352|ID PANCAC_06470;Name 2-oxoglutarate dehydrogenase E1 component |odhA|


### Virulence 

- We found 13 feautes with above 1 log2FoldChange (upregulation in 3h conditions). 

| padj       | log2FoldChange | fullDescription                                                                                                   | gene |
|:---------- | -------------- | ----------------------------------------------------------------------------------------------------------------- | ---- |
|9.50377248836591e-18|3.61087630246594|ID PANCAC_12515;Name immunoglobulin-binding protein Sbi |sbi|
|1.1845449487657e-17|2.80919570021742|ID PANCAC_11885;Name Staphylococcal secretory antigen ssaA2 |ssaA2|
|2.2026694555885e-16|1.16094008607297|ID PANCAC_00295;Name HTH-type transcriptional regulator SarS |sarS|
|6.2511150180482e-14|1.56828353103695|ID PANCAC_03275;Name response regulator transcription factor SaeR |saeR|
|4.31927098430475e-12|1.49465114886715|ID PANCAC_03270;Name two-component system sensor histidine kinase SaeS |saeS|
|9.73601348908573e-11|1.33969408646367|ID PANCAC_03085;Name HTH-type transcriptional regulator SarX |sarX|
|1.73807976163485e-10|1.11464156278183|ID PANCAC_06215;Name Regulatory protein MsrR |msrR|
|2.9975486291052e-09|2.01939285446104|ID PANCAC_13610;Name Clumping factor B%2C adhesin |clfB|
|5.08367236542769e-07|1.27739294778085|ID PANCAC_02505;Name MSCRAMM family adhesin SdrD |sdrD|
|6.01380153103385e-07|1.08881298682941|ID PANCAC_03670;Name phosphopyruvate hydratase |eno|
|2.84281406376053e-05|2.74604090305653|ID PANCAC_10030;Name chemotaxis-inhibiting protein CHIPS |chp|
|0.00138498735621314|1.03699242511397|ID PANCAC_01155;Name type VII secretion system protein EsaA |esaA|
|0.00196546130291416|1.04719886699317|ID PANCAC_01175;Name type VII secretion system protein EssC%2C FtsK/SpoIIIE family ATPase |essC|
|0.0996782650294182|1.22038666830577|ID PANCAC_01185;Name WXG100 family type VII secretion effector EsxB|esxB|


- We found 26 features that are downregulated with below -1 log2FoldChange (in 3h conditions)

| padj       | log2FoldChange | fullDescription                                                                                                   | gene |
|:---------- | -------------- | ----------------------------------------------------------------------------------------------------------------- | ---- |
|2.66608208134608e-78|-2.06986139487564|ID PANCAC_01645;Name alkyl hydroperoxide reductase subunit C |ahpC|
|7.50723907874983e-48|-6.19910290462202|ID PANCAC_01305;Name Na+/proline symporter |putP|
|3.55241198573985e-19|-1.80945016747105|ID PANCAC_05105;Name formyl peptide receptor-like 1 inhibitory protein |flr|
|1.69069204589083e-17|-1.33976285195243|ID PANCAC_09915;Name 4%2C4'-diaponeurosporen-aldehyde dehydrogenase;|aldH1|
|4.62855516794682e-16|-2.04462503432597|ID PANCAC_13280;Name 4%2C4'-diaponeurosporene oxygenase;|crtP|
|2.54469195358708e-11|-2.61271378893334|ID PANCAC_10455;Name delta-hemolysin |hld|
|7.13285606241936e-10|-3.21021086152564|ID PANCAC_01855;Name Phenol-soluble modulin alpha 3 peptide |psmA3|
|1.22138034175356e-09|-3.20475571522958|ID PANCAC_01860;Name Phenol-soluble modulin alpha 2 peptide |psmA2|
|1.54547197782553e-09|-3.1515400154447|ID PANCAC_01850;Name Phenol-soluble modulin alpha 4 peptide |psmA4|
|2.21559520800562e-09|-1.1941178049004|ID PANCAC_08165;Name Sensor histidine kinase WalK |walK|
|5.87154535128768e-09|-1.60678372642257|ID PANCAC_09250;Name peroxide-responsive transcriptional repressor PerR |perR|
|1.00091254263105e-08|-3.14499136253816|ID PANCAC_01865;Name Phenol-soluble modulin alpha 1 peptide |psmA1|
|1.8390394463396e-07|-2.27061111331872|ID PANCAC_03725;Name MSCRAMM family adhesin clumping factor ClfA |clfA|
|3.18142115897223e-07|-1.2563901639856|ID PANCAC_10710;Name Transcriptional regulatory protein KdpE |kdpE|
|8.79261266257935e-06|-1.39872168281259|ID PANCAC_02320;Name ATP-dependent Clp protease ATP-binding subunit ClpC|clpC|
|1.00638838901656e-05|-1.30615897219147|ID PANCAC_13270;Name 4%2C4'-diapophytoene synthase |crtM|
|5.82124341671886e-05|-1.1191420660049|ID PANCAC_13265;Name 4%2C4'-diapophytoene desaturase (4%2C4'-diaponeurosporene-forming) |crtN|
|8.92959696730866e-05|-1.08884734970705|ID PANCAC_10705;Name Sensor histidine kinase KdpD |kdpD|
|0.000458904288255682|-1.00706168134461|ID PANCAC_00355;Name L-serine kinase SbnI |sbnI|
|0.000604591888560002|-1.10078723497004|ID PANCAC_00350;Name staphyloferrin B biosynthesis decarboxylase SbnH |sbnH|
|0.000706621480128811|-1.56839025123286|ID PANCAC_02315;Name protein arginine kinase |mcsB|
|0.00132686161094917|-1.10826424651832|ID PANCAC_00340;Name 3-(L-alanin-3-ylcarbamoyl)-2-[(2-aminoethylcarbamoyl)methyl]-2-hydroxypropanoate synthase SbnF |sbnF|
|0.00275411481821348|-1.57316097859674|ID PANCAC_00320;Name N-[(2S)-2-amino-2-carboxyethyl]-L-glutamate dehydrogenase SbnB |SbnB|
|0.00424614186819964|-1.20802052830176|ID PANCAC_00335;Name L-2%2C3-diaminopropanoate--citrate ligase SbnE |sbnE|
|0.00574071387654988|-1.3156695416079|ID PANCAC_00330;Name staphyloferrin B export MFS transporter |sbnD|
|0.0109223238141383|-1.59341854538431|ID PANCAC_00325;Name staphyloferrin B biosynthesis protein SbnC |sbnC|
|0.190613644205366|-1.14312992931618|ID PANCAC_00315;Name 2%2C3-diaminopropionate biosynthesis protein SbnA|sbnA|

## Top 20 most differently expressed genes 

### Upregulated (in 3h conditions)
| padj       | log2FoldChange | fullDescription                                                                                                   | gene |
|:---------- | -------------- | ----------------------------------------------------------------------------------------------------------------- | ---- |
|1.3194055966605e-104|6.96056454419681|ID PANCAC_03235;Name DNA-binding transcriptional regulator of sugar metabolism%2C DeoR/GlpR family |glpR|
|8.60575730190432e-85|6.1344979982769|ID PANCAC_03240;Name 1-phosphofructokinase |pfkB|
|1.59794180930674e-55|5.17835741484497|ID PANCAC_03245;Name Phosphotransferase system mannitol/fructose-specific IIA domain (Ntr-type) |ptsN|
|1.97679300385683e-17|5.00922261593458|ID PANCAC_12405;Name nitrate reductase subunit alpha |narZ|
|1.22216771362993e-15|4.62658715829128|ID PANCAC_12355;Name putative nitrate transporter NarT |narT|
|4.37128769947428e-16|4.57768803643873|ID PANCAC_12400;Name nitrate reductase subunit beta |narH|
|6.69812311290724e-39|4.43896332021159|ID PANCAC_04125;Name argininosuccinate synthase |argG|
|6.68285306937039e-15|4.09994604941768|ID PANCAC_04525;Name Uncharacterized membrane protein YphA%2C DoxX/SURF4 family |doxX|
|1.36342457159459e-15|4.06969790324893|ID PANCAC_05350;Name Xanthine/uracil permease |uraA|
|4.23945431881971e-12|4.06447640166039|ID PANCAC_12395;Name nitrate reductase molybdenum cofactor assembly chaperone |narJ|
|8.52343445370503e-29|3.84103181598975|ID PANCAC_04120;Name argininosuccinate lyase |argH|
|4.35931954806935e-11|3.75280245873002|ID PANCAC_06610;Name alanine dehydrogenase| ald|
|5.93752035769137e-60|3.64602371134239|ID PANCAC_00825;Name Uncharacterized conserved protein YpbQ%2C isoprenylcysteine carboxyl methyltransferase (ICMT) family |ypbQ|
|9.50377248836591e-18|3.61087630246594|ID PANCAC_12515;Name immunoglobulin-binding protein Sbi |sbi|
|2.83463522421697e-18|3.58352575379707|ID PANCAC_05100;Name complement convertase inhibitor Ecb |ecb|
|6.85185469994296e-13|3.48205223249931|ID PANCAC_00085;Name adenylosuccinate synthase |purA|
|1.27340304117554e-61|3.39788381124344|ID PANCAC_11235;Name Spore dipicolinate transporter SpoVV/YjiH/YlbJ%2C contains nucleoside recognition GATE domain |spoVV|
|3.1008460300964e-11|3.37739879421532|ID PANCAC_05355;Name aspartate carbamoyltransferase catalytic subunit |pyrB|
|3.06052672170772e-05|3.31515345902439|ID PANCAC_12415;Name Ferredoxin subunit of nitrite reductase or a ring-hydroxylating dioxygenase| nirD|
|4.05541740612218e-31|3.28556175992226|ID PANCAC_13155;Name holin-like murein hydrolase modulator CidA| cidA|

### Downregulated (in 3h conditions)
| padj       | log2FoldChange | fullDescription                                                                                                   | gene |
|:---------- | -------------- | ----------------------------------------------------------------------------------------------------------------- | ---- |
|2.6839106111938e-122|-9.90378228766314|ID PANCAC_00885;Name Acyl-CoA dehydrogenase related to the alkylation response protein AidB |caiA|
|3.07613553491979e-222|-8.23753270827405|ID PANCAC_12060;Name urocanate hydratase |hutU|
|1.67753182363656e-160|-8.16860789231566|ID PANCAC_00890;Name O-succinylbenzoic acid-CoA ligase MenE or related acyl-CoA synthetase (AMP-forming) |fadK|
|3.30691017399192e-161|-7.83462963880114|ID PANCAC_00895;Name Acyl CoA:acetate/3-ketoacid CoA transferase |ydiF|
|4.06037167016294e-273|-7.62963956269282|ID PANCAC_12055;Name imidazolonepropionase |hutI|
|1.56539282213039e-63|-7.52126218062103|ID PANCAC_13815;Name YSIRK domain-containing triacylglycerol lipase Lip1 |lip1|
|3.91067181091159e-180|-7.39604422290974|ID PANCAC_00880;Name 3-hydroxyacyl-CoA dehydrogenase;|fadB|
|4.69653211551595e-122|-6.83142874187912|ID PANCAC_00590;Name Putative aldehyde dehydrogenase AldA |aldA|
|2.64772171066377e-163|-6.6371765842846|ID PANCAC_00875;Name Acetyl-CoA acetyltransferase |paaJ|
|9.32969030713222e-107|-6.46004894511155|ID PANCAC_08555;Name Proline dehydrogenase;putA|
|3.89328035738995e-44|-6.3584139366525|ID PANCAC_01310;Name N-acetylneuraminate lyase |nanA|
|9.08892323692643e-164|-6.33077603856762|ID PANCAC_08380;Name acetate--CoA ligase |acsA|
|3.34977417506627e-24|-6.31941590964883|ID PANCAC_01395;Name Transcriptional antiterminator |bglG|
|9.88645215329307e-24|-6.28447308612792|ID PANCAC_01390;Name Phosphotransferase system mannitol/fructose-specific IIA domain (Ntr-type) |ptsN|
|7.50723907874983e-48|-6.19910290462202|ID PANCAC_01305;Name Na+/proline symporter |putP|
|1.23460962893638e-136|-6.09792792162339|ID PANCAC_12625;Name Amino acid permease||
|9.82979616471251e-135|-6.07889826538479|ID PANCAC_08680;Name phosphoenolpyruvate carboxykinase (ATP) |pckA|
|1.28305405688087e-45|-6.03022216126546|ID PANCAC_12560;Name dethiobiotin synthase |bioD|
|1.88263245292698e-31|-6.0000900578587|ID PANCAC_00635;Name NAD-dependent formate dehydrogenase|ldhA|
|7.88877624806523e-59|-5.97888711361208|ID PANCAC_00705;Name 6-phospho-N-acetylmuramidase|mupG|

## COG
Selected from padj < 0.05
Legend: 
| Letter | Description                                                       |
|--------|-------------------------------------------------------------------|
| D      | Cell cycle control, cell division, chromosome partitioning        |
| M      | Cell wall/membrane/envelope biogenesis                            |
| N      | Cell motility                                                     |
| O      | Post-translational modification, protein turnover, and chaperones |
| T      | Signal transduction mechanisms                                    |
| U      | Intracellular trafficking, secretion, and vesicular transport     |
| V      | Defense mechanisms                                                |
| W      | Extracellular structures                                           |
| Y      | Nuclear structure                                                 |
| Z      | Cytoskeleton                                                      |
| A      | RNA processing and modification                                   |
| B      | Chromatin structure and dynamics                                  |
| J      | Translation, ribosomal structure and biogenesis                   |
| K      | Transcription                                                     |
| L      | Replication, recombination and repair                             |
| C      | Energy production and conversion                                  |
| E      | Amino acid transport and metabolism                               |
| F      | Nucleotide transport and metabolism                               |
| G      | Carbohydrate transport and metabolism                             |
| H      | Coenzyme transport and metabolism                                 |
| I      | Lipid transport and metabolism                                    |
| P      | Inorganic ion transport and metabolism                            |
| Q      | Secondary metabolites biosynthesis, transport, and catabolism     |
| R      | General function prediction only                                  |
| S      | Function unknown                                                  |

### Upregulated

|              |      |
| ------------ | ---- |
| Assigned COG | 266      |
| NA           | 54 |
![cog_up_time](https://hackmd.io/_uploads/HyJ_C-PA6.jpg)




### Downregulated

|              |      |
| ------------ | ---- |
| Assigned COG | 363      |
| NA           | 96 |
![cog_down_time](https://hackmd.io/_uploads/H1DcCZvA6.jpg)




