AdamHuckaby / guler-dna-pipeline

DNA processing pipeline for Jennfier Guler's Biology Lab at UVA

guler-dna-pipeline

DNA processing pipeline for Jennfier Guler's Biology Lab at UVA

See dependencies.md for installation instructions. Repo supports 64-bit Linux and MacOS only.

Task List

• Import DNA sequence reads and create consistent name conventions. Currently ~150 genomes in fastq format. report_gen.R is our current best attempt at this. Works in sample fastq.gz files in this repository, but not all we have access to. The fast.qz files are stored on Adam's machine only at the moment.

• Quality control on reads, FastQC.

• Align reads using BWA-MEM to create sam/bam files. Recommended settings command is: bwa mem ref.fa read1.fq read2.fq > aln-pe.sam

• Use CNVnator to analyze bam files

• Use Delly2 to analyze bam files

• Compare CNV breakpoint locations from CNVnator to locations from Delly2 and extract sequences at breakpoints for further analysis

Collecting Data from fastq.gz Files

Given clinical_data/ directory containing .fastq.gz files:
Rscript report_gen.R ~/Desktop/guler-lab/clinical_data/

Make sure to use the absolute path to the directory.

###download DSM1 files using SRA, may have to change the path for your installation of sratoolkit /home/ahuckaby/sratoolkit.2.8.2-1-centos_linux64/bin/fastq-dump --split-files SRR941237 /home/ahuckaby/sratoolkit.2.8.2-1-centos_linux64/bin/fastq-dump --split-files SRR941239 /home/ahuckaby/sratoolkit.2.8.2-1-centos_linux64/bin/fastq-dump --split-files SRR941241

###rename 37 to D731, 39 to Dd2, and 41 to C710 ###replace all ".1 " with "/1 " etc sed -i -- 's.1 /1 \g' * sed -i -- 's.2 /2 \g' * sed -i -- 's.3 /2 \g' *

###interleave reads, trim white space, and compress reformat.sh in=Dd2_1.fastq in2=Dd2_2.fastq out=Dd2_interleaved.fastq.gz trd reformat.sh in=C710_1.fastq in2=C710_2.fastq out=C710_interleaved.fastq.gz trd reformat.sh in=D731_1.fastq in2=D731_2.fastq out=D731_interleaved.fastq.gz trd

###run QC on reads as necessary using bbtools

##use speedseq to align, could add in qualifiers to keep names consistent and run all at once in the future speedseq align -p -v -t 4 -o Dd2align -M 3 -R "@RG\tID:Dd2.s1\tSM:CGATGT\tLB:lib1" /home/ahuckaby/genomes/Pfal/PlasmoDB-31_Pfalciparum3D7_Genome.fasta /home/ahuckaby/Desktop/DSM1/Dd2_interleaved.fastq.gz

speedseq align -p -v -t 4 -o C710align -M 3 -R "@RG\tID:C710.s1\tSM:CGATGT\tLB:lib2" /home/ahuckaby/genomes/Pfal/PlasmoDB-31_Pfalciparum3D7_Genome.fasta /home/ahuckaby/Desktop/DSM1/C710_interleaved.fastq.gz

speedseq align -p -v -t 4 -o D731test -M 3 -R "@RG\tID:D731a.s1\tSM:CGATGT\tLB:lib3" /home/ahuckaby/genomes/Pfal/PlasmoDB-31_Pfalciparum3D7_Genome.fasta /home/ahuckaby/Desktop/DSM1/D731_interleaved.fastq.gz