# Squire test on Drosophila samples
###### tags: `TE_expression` `RNAseq` `dm6`

## Dataset

Two RNAseq libraries from Marie Fablet.

### FastQC & Trimming

Paired-end 100bp

| Sample Name | M Seqs | Trimmed |
|:------------- | ------ |:-------:|
| CNVR359_S2.R1 | 24.7 | 18.5 |
| CNVR359_S2.R2 | 24.7 | 18.5 |
| CNVR376_S2.R1 | 17.4 | 13.5 |
| CNVR376_S2.R2 | 17.4 | 13.5 |

Trimming
Didn't bother with adaptor trimming. If needed, use trim_galore instead.

while read line; 
do  
 fileNameR1=$(echo $line|awk '{print $1}'); 
 fileNameR2=$(echo $line|awk '{print $2}'); 

#trimming
java -jar  /home/data/rrebollo/Trimmomatic-0.39/trimmomatic-0.39.jar PE -threads 4 -phred33 $fileNameR1.fastq.gz $fileNameR2.fastq.gz $fileNameR1.paired.fastq $fileNameR1.unpaired.fastq.gz $fileNameR2.paired.fastq $fileNameR2.unpaired.fastq.gz LEADING:20 TRAILING:20 AVGQUAL:25 SLIDINGWINDOW:10:30 MINLEN:35;

done<marie_samples.txt

## Squire run on paired-end sequences

Github : https://github.com/wyang17/SQuIRE

Need to change the count script as seen here : https://github.com/wyang17/SQuIRE/issues/19

#install squire
#create conda env with 2.7.3a STAR
conda create --name squire --override-channels -c iuc -c bioconda -c conda-forge -c defaults -c r python=2.7.13 bioconductor-deseq2=1.16.1 r-base=3.4.1 r-pheatmap bioconductor-vsn bioconductor-biocparallel=1.12.0 r-ggrepel star=2.7.3a bedtools=2.25.0 samtools=1.1 stringtie=1.3.3 igvtools=2.3.93 ucsc-genepredtobed ucsc-gtftogenepred ucsc-genepredtogtf ucsc-bedgraphtobigwig r-hexbin
#activate environment
source activate squire
#install squire
git clone https://github.com/wyang17/SQuIRE; cd SQuIRE; pip install -e .

#Squire
squire Fetch -b dm6 -f -c -r -g -x -p 4 -v
squire Clean -b dm6 -v
squire Map -b dm6 -g -p 4 -r 100 -1  CNVR359_S2.R1.paired.fastq -2 CNVR359_S2.R2.paired.fastq -v
squire Count -b dm6 -r 100 -n CNVR359 -p 4 -s 1 -v
squire Map -b dm6 -g -p 4 -r 100 -1  CNVR376_S2.R1.paired.fastq -2 CNVR376_S2.R2.paired.fastq -v
squire Count -b dm6 -r 100 -n CNVR376 -p 4 -s 1 -v
squire Call -1 CNVR359 -2 CNVR376 -A CNVR376 -B CNVR376 -p 4 -N Droso_Marie -f pdf -v 

*Marie* you can check the multiqc on the FTP to see all the mapping statistics. Don't bother with the samples "single".

## Squire run on single-end sequences

Ran squire on R1 as single-end in order to have a similar run as our DualRNAseq data.

#Squire
squire Map -b dm6 -g -p 4 -r 100 -1  CNVR359_S2.R1.single.fastq -v
squire Count -b dm6 -r 100 -n CNVR359_S2.R1.single -p 4 -s 1 -v
squire Map -b dm6 -g -p 4 -r 100 -1  CNVR376_S2.R1.single.fastq -v
squire Count -b dm6 -r 100 -n CNVR376_S2.R1.single -p 4 -s 1 -v

## Data on the FTP

All samples labeled "paired" stem from the paired end analysis, the "single" from the single end analysis. The raw data and trimmed data is present in the Test_Marie_Squire folder. Within are all squire folders, created automatically by squire:

* Squire fetch : genome + gene and TE annotations + STAR index
* Squire clean : TE annotation simplified (automatically done by squire clean)
* Squire map: BAM files (squire uses STAR) and all STAR info
* Squire count: count table per TE copy + gene count table
* Squire call : deseq2 on copies