Data Analysis of Mutation Detection in Gamma-Irradiated Banana Using Low Copy Number Variation

Software Prerequisites

#Burrows-Wheeler-Aligner (http://bio-bwa.sourceforge.net/)(see line 126). #Download and Install BBmap https://sourceforge.net/projects/bbmap/ Bin-by-Sam-tool (see github repository) Python version 2.7(See enivornment .yaml)

Banana

Procure your raw FASTQ reads from NCBI of two Banana samples, one is a known mutant Novaria and the other is a wildtype Naine and follow the protocol. Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana p.117 - 127 Use clumpify script to remove duplicates

(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA627139)

#Download sratools

srapath SRR11579627

prefetch SRR11579627

wget https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-run-21/SRR11579627/SRR11579627.1

#Convert SRA into fastq

fastq-dump --split-3 SRR11579627


srapath SRR11579628

prefetch SRR11579628

wget https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos3/sra-pub-run-21/SRR11579628/SRR11579628.1

#Convert SRA into fastq

fastq-dump --split-3 SRR11579628

Rename FASTQ

Naine.R1.fq.gz Naine.R2.fq.gz Novaria.R1.fq.gz Novaria.R2.fq.gz

Run the clumpify python script to remove duplicates per sample.

./clumpify.sh in=Naine.R1.fq.gz in2=Naine.R2.fq.gz out=Naine.R1.
dedup.fastq.gz out2=Naine.R2.dedup.fastq.gz dedupe=t

./clumpify.sh in=Novaria.R1.fq.gz in2=Novaria.R2.fq.gz out=Novaria.R1.
dedup.fastq.gz out2=Novaria.R2.dedup.fastq.gz dedupe=t

Standard Output Clumpify python

Done! Time: 31.447 seconds. Reads Processed: 6262k 199.16k reads/sec Bases Processed: 1885m 59.94m bases/sec

Reads In: 6262958 Clumps Formed: 1730359 Duplicates Found: 3782 Reads Out: 6259176 Bases Out: 1884185686 Total time: 51.345 seconds.

NOVARIA

Done! Time: 29.438 seconds. Reads Processed: 6000k 203.82k reads/sec Bases Processed: 1837m 62.43m bases/sec

Reads In: 6000036 Clumps Formed: 1648176 Duplicates Found: 2026 Reads Out: 5998010 Bases Out: 1837286910 Total time: 50.222 seconds.

Download Reference Genome NCBI

https://www.ncbi.nlm.nih.gov/assembly/GCF_000313855.2

mkdir BananaGamma
mv Novaria.R1.dedup.fastq.gz Novaria.R2.dedup.fastq.gz BananaGamma/
mv Naine.R1.dedup.fastq.gz Naine.R2.dedup.fastq.gz BananaGamma/
cd BananaGamma

mkdir Genome
mv *.fna Genome/
cd Genome bwa index *.fna

cd ../

https://github.com/lh3/bwa

#It is recommended not to download bwa software from source a.k.a github but rather through bioconda channel in conda
#conda install -c bioconda bwa=0.7.17

#Another way to get bwa software but do not necessarily use it.
git clone https://github.com/lh3/bwa.git
cd bwa; make
./bwa
#Needs to be Harvard Version

./bwa mem -M -t 4 -R '@RG\tID:Novaria \tSM: Novaria' ../Genome/*.fna Novaria.R1.dedup.fq Novaria.R2.dedup.fq > Novaria.dedup.sam

./bwa mem -M -t 4 -R '@RG\tID:Naine \tSM: Naine' ../Genome/*.fna Naine.R1.dedup.fastq.gz Naine.R2.dedup.fastq.gz > Naine.dedup.sam

samtools sort -O sam -T sam -T Novaria.sort -o Novaria_aln.sam Novaria.dedup.sam
samtools sort -O sam -T sam -T Naine.sort -o Naine_aln.sam Naine.dedup.sam

samtools view -b Novaria_aln.sam > Novaria.bam
samtools view -b Naine_aln.sam > Naine.bam

samtools index Novaria.bam
samtools index Naine.bam

mv Novaria_aln.sam Naine_aln.sam Bin-by-Sam-tool/
cd Bin-by-Sam-tool
python bin-by-sam_2.0.py -o N3_100kbin.txt -s 100000 -b -p 3 -c Naine_.aln.sam

Download the r package rom PBGLMichael/CNVseq repository

# Banana CNV

setwd("/home/michael/Desktop/Banana/Banana_LC_WGS")
devtools::install_github(repo = "PBGLMichaelHall/CNVseq",force = TRUE)
library(CNV)

CNV::CNV(file = "N3_100kbin.txt",Chromosome =  c("NC_025202.1","NC_025203.1","NC_025203.1","NC_025204.1","NC_025205.1",
"NC_025206.1","NC_025207.1","NC_025208.1","NC_025209.1","NC_025210.1","NC_025211.1","NC_025212.1"),
mutantname = "Novaria.Naine",controlname = "Naine.Naine",size = .75,alpha = 5,color="orange")
_images/Rplot02.png
_images/BANANA100K.png

Chromosome 5

CNV::CNV(file = "N3_100kbin.txt",Chromosome =  c("NC_025206.1"),mutantname = "Novaria.Naine",
controlname = "Naine.Naine",size = .75,alpha = .25,color="green")
_images/Rplot03.png

Sorghum CNV

**You have two BAM files one is a "mutant" and the other is a "control"** **First convert BAM to SAM** **The sam file must have an ending _aln.sam to work properly in python script** **CONTROL**
samtools view -h con-2_S1-Chromes-04-05-09.bam > con-2_S1-Chromes-04-05-09_aln.sam

**MUTANT**

samtools view -h D2-1_S7-Chromes-04-05-09.bam > D2-1_S7-Chromes-04-05-09_aln.sam

bin-by-sam_2.0.py python script

$python bin-by-sam_2.0.py -o N3_100kbin.txt -s 100000 -b -p 2 -c con-2_S1-Chromes-04-05-09_aln.sam

CNV::CNV(file = "N3_100kbin.txt",Chromosome = c("Chr04","Chr05","Chr09"),mutantname = "D2.1_S7.Chromes.04.05.09.con.2_S1.Chromes.04.05.09",controlname = "con.2_S1.Chromes.04.05.09.con.2_S1.Chromes.04.05.09",size = .75,alpha = 5.0,color="orange")
_images/Rplot99.png

Chromosome 9

CNV::CNV(file = "N3_100kbin.txt",Chromosome = c("Chr09"),mutantname = "D2.1_S7.Chromes.04.05.09.con.2_S1.Chromes.04.05.09",controlname = "con.2_S1.Chromes.04.05.09.con.2_S1.Chromes.04.05.09",size = .75,alpha = 5.0,color="orange")
_images/Rplot.png