Last updated: 2021-02-15
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Knit directory: Mouse_AAV_PGR_RNAseq_bulk/
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Following sequencing and obtaining .fastq.gz file, the first step is to perform trimming and mapping of the sequencing data to generate bam files. All these steps were performed using bash code.
Bam files were then used for read counts to generate a count matrix.
Mouse AAV-PGR bulk RNA-seq were performed using paiered end sequencing method and below are the scripts for trimming and mapping paired end sequencing read.
#!/bin/bash
# function to run skewer quality trimming
runskew(){
FQZ1=$1
FQZ2=`echo $FQZ1 | sed 's/_R1.fastq.gz/_R2.fastq.gz/'`
skewer -t 8 -q 20 $FQZ1 $FQZ2
}
export -f runskew
# actually run skewer
parallel -j3 runskew ::: *_R1.fastq.gzIt will generate the following 4 outputs for individual .fastq.gz file:
#!/bin/bash
DIR=/group/card2/Evangelyn_Sim/Sequencing_ATAC_RNA/refgenome/star
GTF=/group/card2/Evangelyn_Sim/Sequencing_ATAC_RNA/refgenome/Mus_musculus.GRCm38.96.gtf
for FQ1 in `ls *1.fastq-trimmed-pair1.fastq` ; do
FQ2=`echo $FQ1 | sed 's/1.fastq-trimmed-pair1.fastq/1.fastq-trimmed-pair2.fastq/'`
BASE=`echo $FQ1 | sed 's/_1.fastq-trimmed-pair1.fastq//'`
STAR --genomeLoad NoSharedMemory --genomeDir $DIR --readFilesIn $FQ1 $FQ2 --runThreadN 30 \
--sjdbGTFfile $GTF --outSAMattributes NH HI NM MD
rm $FQ1 $FQ2
mv Aligned.out.sam ${BASE}.STAR.sam
mv Log.final.out ${BASE}_starlog.txt
( samtools view -uSh ${BASE}.STAR.sam | samtools sort -o ${BASE}.STAR.bam
rm ${BASE}.STAR.sam
samtools index ${BASE}.STAR.bam
samtools flagstat ${BASE}.STAR.bam > ${BASE}.STAR.bam.stats ) &
done
STAR genomeLoad Remove --genomeDir $DIR
wait
ls: cannot access *1.fastq-trimmed-pair1.fastq: No such file or directory
bash: line 24: STAR: command not foundMake a directory called “merged” and ln all .bam files to the folder and perform the following.
#!/bin/bash
samtools view -H `ls *bam | head -1` > header.sam
for BASE in `ls *bam | cut -d '_' -f2 | sort -u ` ; do
rm $BASE.mg.bam
samtools merge -h header.sam $BASE.mg.bam *${BASE}*bam &
done
wait
ls: cannot access *bam: No such file or directory
bash: line 2: samtools: command not found
ls: cannot access *bam: No such file or directory
#!/bin/bash
SAF=/group/card2/Evangelyn_Sim/Sequencing_ATAC_RNA/refgenome/Mus_musculus.GRCm38.96.fulllength.saf
OUT=mrna_fulllen_pe_strrev_q30.mx
#featureCounts -p -Q 30 -T 20 -s 2 -a $SAF -F SAF -o $OUT *bam
#!/bin/bash
for MX in `ls *mx` ; do
sed 1d $MX | sed 's/.mg.bam//g' > $MX.all
sed 1d $MX | cut -f1-6 | sed 's/.mg.bam//g' > $MX.chr
sed 1d $MX | cut -f1,7- | sed 's/.mg.bam//g' > $MX.PR.fix
done
wait
ls: cannot access *mx: No such file or directoryFiltering out low counts genes by running the following filter.sh as
bash filter.sh hrna_dev_mf_fulllen_se_strrev_q30.mx.all.fix
filter.sh
head -1 $1 > ${1}_filt
awk '{
min = max = sum = $2; # Initialize to the first value (2nd field)
sum2 = $2 * $2 # Running sum of squares
for (n=3; n <= NF; n++) { # Process each value on the line
if ($n < min) min = $n # Current minimum
if ($n > max) max = $n # Current maximum
sum += $n; # Running sum of values
sum2 += $n * $n # Running sum of squares
}
print sum/(NF-1) ;
}' $1 > avg
paste avg $1 | awk '$1 >= 10' | cut -f2- | tr ' ' '\t' >> ${1}_filt
rm avgTo confirm the expression of AAV-PGR (human isoform), Skewer trimmed reads also mapped to human reference genome and later counted.
#!/bin/bash
DIR=/group/card2/Evangelyn_Sim/Transcriptome_chromatin_human/Sequencing_ATAC_RNA/refgenome/star
GTF=/group/card2/Evangelyn_Sim/Transcriptome_chromatin_human/Sequencing_ATAC_RNA/refgenome/star/Homo_sapiens.GRCh38.96.gtf
for FQ1 in `ls *1.fastq-trimmed-pair1.fastq` ; do
FQ2=`echo $FQ1 | sed 's/1.fastq-trimmed-pair1.fastq/1.fastq-trimmed-pair2.fastq/'`
BASE=`echo $FQ1 | sed 's/_1.fastq-trimmed-pair1.fastq//'`
STAR --genomeLoad NoSharedMemory --genomeDir $DIR --readFilesIn $FQ1 $FQ2 --runThreadN 30 \
--sjdbGTFfile $GTF --outSAMattributes NH HI NM MD
rm $FQ1 $FQ2
mv Aligned.out.sam ${BASE}.STAR.sam
mv Log.final.out ${BASE}_starlog.txt
( samtools view -uSh ${BASE}.STAR.sam | samtools sort -o ${BASE}.STAR.bam
rm ${BASE}.STAR.sam
samtools index ${BASE}.STAR.bam
samtools flagstat ${BASE}.STAR.bam > ${BASE}.STAR.bam.stats ) &
done
STAR genomeLoad Remove --genomeDir $DIR
wait
ls: cannot access *1.fastq-trimmed-pair1.fastq: No such file or directory
bash: line 22: STAR: command not found
#!/bin/bash
SAF=/group/card2/Evangelyn_Sim/Transcriptome_chromatin_human/Sequencing_ATAC_RNA/refgenome/Homo_sapiens.GRCh38.96.fulllength.saf
OUT=mrna_fulllen_pe_strrev_q30_map2human.mx
#featureCounts -p -Q 30 -T 20 -s 2 -a $SAF -F SAF -o $OUT *bam
sessionInfo()R version 3.6.1 (2019-07-05)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)
Matrix products: default
BLAS: /hpc/software/installed/R/3.6.1/lib64/R/lib/libRblas.so
LAPACK: /hpc/software/installed/R/3.6.1/lib64/R/lib/libRlapack.so
locale:
[1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
[3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
[5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] workflowr_1.6.2
loaded via a namespace (and not attached):
[1] Rcpp_1.0.5 rstudioapi_0.11 whisker_0.4 knitr_1.30
[5] magrittr_1.5 R6_2.5.0 rlang_0.4.7 stringr_1.4.0
[9] tools_3.6.1 xfun_0.18 git2r_0.27.1 htmltools_0.5.0
[13] ellipsis_0.3.1 rprojroot_1.3-2 yaml_2.2.1 digest_0.6.27
[17] tibble_3.0.3 lifecycle_0.2.0 crayon_1.3.4 later_1.1.0.1
[21] vctrs_0.3.2 promises_1.1.1 fs_1.5.0 glue_1.4.2
[25] evaluate_0.14 rmarkdown_2.5 stringi_1.5.3 compiler_3.6.1
[29] pillar_1.4.6 backports_1.1.10 httpuv_1.5.4 pkgconfig_2.0.3