---
# System prepended metadata

title: processing of test data
tags: [' STAR', QC]

---

---
tags: QC, STAR, filename-editing, slurm-script
--- 

# processing of test data

12April22

1. Downloaded read data and put in `/mnt/lustre/macmaneslab/macmanes/physiology/reads`

2. edited read file names to remove `001`

```
for filename in *fastq.gz; do                  
    [ -f "$filename" ] || continue
    mv "$filename" "${filename//_001/}"
done
```

3. put genome and gff in `/mnt/lustre/macmaneslab/macmanes/physiology/genome`

4. Indexed genome using STAR. The script is in `$HOME/physiology` and named `star_gg.slurm`

```
#!/bin/bash

#SBATCH --partition=macmanes
#SBATCH -J star_gg
#SBATCH --cpus-per-task=40
#SBATCH --output star.log

source ~/.bash_profile


mkdir $HOME/physiology/STAR_indices

STAR --runMode genomeGenerate --runThreadN 40 --genomeDir $HOME/physiologyc/STAR_indices \
--genomeFastaFiles $HOME/physiology/genome/Peer2.0.1.fasta \
--sjdbGTFfile $HOME/physiology/genome/Peromyscus_eremicus__Peer2.0.1.fasta_v3.functional.gff3
```

5. moved the index files to `$HOME/physiology/STAR_index`
6. Did the mapping job. The script is in `$HOME/physiology` and is named `ReadCount.job`. It is executed like this

```
sbatch $HOME/ReadCount.job \
/mnt/lustre/macmaneslab/macmanes/physiology/genome/Peer2.0.1.fasta \
/mnt/lustre/macmaneslab/macmanes/physiology/genome/Peromyscus_eremicus__Peer2.0.1.fasta_v3.functional.gff3 \
/mnt/lustre/macmaneslab/macmanes/physiology/reads/first/pilot/220408_A01346_0055_AH22JTDRX2_16MerMACMANES_RNA_PILOT \
.fastq.gz
```

the actual mapping command is 

```
STAR --runMode alignReads \
--genomeDir $DIR/STAR_index \
--readFilesIn ${READ_DIR}/${sample}R1${SUFFIX} ${READ_DIR}/${sample}R2${SUFFIX} \
--readFilesCommand zcat \
--runThreadN 40 \
--outFilterMultimapNmax 20 \
--outFilterMismatchNmax 10 \
--outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0.3 \
--quantMode GeneCounts \
--outFileNamePrefix ${DIR}/STAR_mapped/${sample} \
--outSAMtype BAM Unsorted \
--genomeLoad LoadAndKeep

```

7. once the mapping is done, I pull out all the mapping data using the following command to make a nice table.

```
printf "%s\t%s\t%s\t%s\t%s\t%s\t%s\n" "date" "sample" "reads" "unique" "multi" "toomany" "unmapped"> mappingdata.txt
for file in $(lr *Log.final.out | awk '{print $9}')
do
sample=$(echo $file | sed "s/_Log.final.out//g" | sed "s/.Log.final.out//g")
date=$(grep "Started job" $file | cut -f 2 | cut -d ' ' -f1-2)
reads=$(grep "Number of input reads" $file | cut -f 2)
unique=$(grep "Uniquely mapped reads %" $file | cut -f 2)
multi=$(grep "% of reads mapped to multiple loci" $file | cut -f 2)
toomany=$(grep "% of reads mapped to too many loci" $file | cut -f 2)
unmapped=$(grep "Number of reads unmapped: too short" $file | cut -f 2)
printf "%s\t%s\t%s\t%s\t%s\t%s\t%s\n" "$date" "$sample" "$reads" "$unique" "$multi" "$toomany" "$unmapped" >> mappingdata.txt
done
```

it looks like this

```
Sample  Number_reads    Uniquely_mapping_reads  Mapped_to_multiple_loci Mapped_to_too_many_loci Unmapped_reads_too_short
P1_A10_S73_L001_        741096  77.05%  22.13%  0.08%   4776
P1_A11_S81_L001_        360614  80.69%  18.40%  0.08%   2577
P1_A12_S89_L001_        567409  78.29%  20.82%  0.08%   4215
P1_A1_S1_L001_  589599  80.62%  18.48%  0.11%   3931
P1_A2_S9_L001_  715087  77.35%  21.82%  0.08%   4616
P1_A3_S17_L001_ 478268  80.11%  18.84%  0.08%   4150
P1_A4_S25_L001_ 504477  79.56%  19.46%  0.09%   3866
P1_A5_S33_L001_ 263045  78.02%  21.18%  0.08%   1674
P1_A6_S41_L001_ 258891  79.53%  18.74%  0.07%   3756
```

On the basis of this experiments, I asked Steve to repool the libraries, bring some concentratons up, some down, and to exclude some. That spreadsheet is [here](https://universitysystemnh-my.sharepoint.com/:x:/g/personal/mdm2000_usnh_edu/EebVFrf5eehAgPnuOVoSUsIBOx1ZiYuYdekPpUtVttrYAw?e=Z57b1S) 

```
sbatch ./ReadCount.job \
/mnt/lustre/macmaneslab/macmanes/physiology/genome/Peer2.0.1.fasta \
/mnt/lustre/macmaneslab/macmanes/physiology/genome/Peromyscus_eremicus__Peer2.0.1.fasta_v3.functional.gff3 \
/mnt/lustre/macmaneslab/macmanes/physiology/raw_reads/second_run/pilot/cobb.sr.unh.edu/managed/220810_A01346_0075_BH5H53DRX2_8MerMACMANES_RNA_P4/reads \
.fastq.gz
```