hello everyone,
I'm using BWA+SAMtools+Varscan to call indels to check my sgRNA cutting efficiency. The protocol I used is shown below:
1> set index: bwa index hg38.fa
2> BWA-mem: bwa mem -M hg38.fa XD01_R1.fastq XD02_R2.fastq > XD01.sam
3> sam 2 bam: samtools view -bS XD01_reorder.sam -o XD01.bam
4> sort bam: samtools sort XD01.bam > XD01.sorted.bam
5> bam index: samtools index XD01_sorted.bam
6> bam 2 mpileup: samtools mpileup -f hg38.fa XD01_sorted.bam > XD01.mpileup
7> mpileup 2 vcf: java -jar VarScan.jar mpileup2indel XD01.mpileup --min-var-freq 0 --p-value 0.99 --min-avg-qual 1 --min-coverage 1 --output-vcf 1 > XD01.vcf
Through these steps I got the vcf file finally. But the number of indels are very small. For example, only 5 indels were found in one of my sam files (2000+ SNPs). I don't know if there is some wrong with my experiment? Or some wrong with my indel calling process?
Hi , Did you try to change your --p-value parameters ?
All the pvalues of the ALT are 0.98. I once tried to change pvalue to 0.01. No ALT was detected under that situation.
May be you can try with --strand-filter 0 but i m not sure it will help.
Have you tried an established pipeline, such as CRISPResso to compare the results? Or try playing with the GAP penalties of bwa mem, maybe your reads get dropped due to low scoring?