# Revision session - Day 4

## Feedback

* Not sure my sense of humour is as Bastian's... so bare with me :sweat_smile:

* Flowchart:

flowchart TD
    data --> FastQC 
    data --> SortMeRNA
    SortMeRNA --> FastQC
    SortMeRNA --> Trimmomatic
    Trimmomatic --> FastQC
    FastQC --> MultiQC
    SortMeRNA --> MultiQC
    Trimmomatic --> MultiQC
    Trimmomatic --> rCorrector
    rCorrector --> Trinity
    Trinity --> Detonate
    Trinity --> Transrate
    Trinity --> BUSCO
    Trinity --> transdecoder
    transdecoder --> eggnog
    Trimmomatic --> salmon
    Trinity --> salmon
    salmon --> DESeq2
    

* eggnog output: we follow the trail from the GitHub [repository](https://github.com/eggnogdb/eggnog-mapper) to the [WIKI](https://github.com/eggnogdb/eggnog-mapper/wiki) to the current version [output description](https://github.com/eggnogdb/eggnog-mapper/wiki)

From `Elsa`:

## Wed Nov 30 20:03:08 2022
## emapper-2.1.9
## /usr/local/bin/eggnog-mapper-2.1.9/emapper.py --cpu 4 -i /home/elza/day3/transcriptome/annotation/decoded/longest_orfs.pep --translate -m diamond -o /home/elza/day3/transcriptome/annotation/eggnog
##
#query  seed_ortholog   evalue  score   eggNOG_OGs  max_annot_lvl   COG_category    Description Preferred_name  GOs EC  KEGG_ko KEGG_Pathway    KEGG_Module KEGG_Reaction   KEGG_rclass BRITE   KEGG_TC CAZy    BiGG_Reaction   PFAMs
TRINITY_DN1_c0_g1_i1.p1 3880.AES99404   2.71e-78    261.0   COG5099@1|root,KOG1488@2759|Eukaryota,37HNU@33090|Viridiplantae,3GAB4@35493|Streptophyta,4JMHX@91835|fabids 35493|Streptophyta  J   pumilio homolog -   GO:0003674,GO:0003676,GO:0003723,GO:0003729,GO:0003730,GO:0005488,GO:0005575,GO:0005622,GO:0005623,GO:0005737,GO:0005829,GO:0044424,GO:0044444,GO:0044464,GO:0097159,GO:1901363 -   ko:K17943   -   -   -   -   ko00000 -   -   -   NABP,PUF
TRINITY_DN1_c0_g1_i2.p1 3880.AES99404   1.08e-92    301.0   COG5099@1|root,KOG1488@2759|Eukaryota,37HNU@33090|Viridiplantae,3GAB4@35493|Streptophyta,4JMHX@91835|fabids 35493|Streptophyta  J   pumilio homolog -   GO:0003674,GO:0003676,GO:0003723,GO:0003729,GO:0003730,GO:0005488,GO:0005575,GO:0005622,GO:0005623,GO:0005737,GO:0005829,GO:0044424,GO:0044444,GO:0044464,GO:0097159,GO:1901363 -   ko:K17943   -   -   -   -   ko00000 -   -   -   NABP,PUF
TRINITY_DN10_c0_g1_i3.p1    13333.ERM97415  3.78e-153   462.0   28HBY@1|root,2QPQB@2759|Eukaryota,37NM8@33090|Viridiplantae,3G8U4@35493|Streptophyta    35493|Streptophyta  S   ETO1-like protein 1 -   -   -   -   -   -   -   -   -   -   -   -   BTB,TPR_8
TRINITY_DN10_c0_g1_i4.p1    13333.ERM97415  3.78e-153   462.0   28HBY@1|root,2QPQB@2759|Eukaryota,37NM8@33090|Viridiplantae,3G8U4@35493|Streptophyta    35493|Streptophyta  S   ETO1-like protein 1 -   -   -   -   -   -   -   -   -   -   -   -   BTB,TPR_8
## 4 queries scanned
## Total time (seconds): 0.9135375022888184
## Rate: 4.38 q/s

## Assessment

1. Oxford Nanopore Direct Sequencing is an amplification free protocol **TRUE**/False

2. Which one is not a step of the trinity short-read de-novo assembly
* **circular consensus sequence**
* linear contig generation (inchworm)
* de Bruijn graph generation (chrysalis)
* de Bruijn graph traversal (butterfly)

The last three are all steps of trinity.

3. List one advantage and one limitation of 3rd generation sequencing over 2nd generation sequencing in the context of de-novo transcriptome assembly.

`3rd gen will do MORE for me! :) ... if I have the $$$$$`

4. Which is NOT a method that could be used to assess an assembly quality?

* **SortMeRNA**
* transrate
* BUSCO
* detonate

Well you got it right, albeit we did not cover that topic yesterday... Why are we there? :-D

5. Describe in ONE sentence the principle of EITHER detonate or BUSCO.

`detonate: the state of my mind after a full day of lectures`

`detonate is so fancy and awesome that it will blow my mind tomorrow :-)`

## Questions?

## A bit more on reproducible research

You have heard about making the data FAIR and Bastian mentioned numerous methods to augment reproducibility (container, conda). You have also seen Nextflow. We still miss one piece to have all the minimal skills. Here it comes:

### GitHub bonus!

1. I will create a public repository for the course
2. You will clone that repository
2. I will create a script to run salmon
3. I will create a script to iterate through the files
4. I will commit and push my changes
5. You will pull the changes
6. You will run the script

[There](https://gist.github.com/nicolasDelhomme/46a1053d277510b95692318bd1732b6d) you will find the Git instructions we provide to onboard user at the UPSC. Most of the information there will be relevant to you if you ever want to use GitHub in your research.

### CLI commands

mkdir salmon
mkdir indices
salmon -h
salmon index -h
salmon quant -h
salmon quant --help-reads

# build the index using salmon index - check the lecture for additional pointers.
salmon index -t /data/raw_data/reference/fasta/Pabies1.0-all-phase.gff3.CDSandLTR-TE.fa.gz  -i indices/Pabies1.0-all-phase.gff3.CDSandLTR-TE_salmon_1.8.0

# iterate on all samples, run salmon
for f in $(find /data/raw_data/trimmomatic -name "*_trimmomatic_1.fq.gz"); do
	fnam=$(basename ${f/_1.fq.gz/})
	salmon quant -l A \
	-i ~/indices/Pabies1.0-all-phase.gff3.CDSandLTR-TE_salmon_1.8.0 \
	-1 $f -2 /data/raw_data/trimmomatic/${fnam}_2.fq.gz \
	-o salmon/$fnam --seqBias --gcBias \
	-p 4
done