Question: Is there something wrong with my Indel Calling protocol?
gravatar for lishen0709
15 months ago by
lishen07090 wrote:

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?

rna-seq bwa alignment genome indel • 537 views
ADD COMMENTlink written 15 months ago by lishen07090

Hi , Did you try to change your --p-value parameters ?

ADD REPLYlink written 15 months ago by Titus810

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.

ADD REPLYlink written 15 months ago by lishen07090

May be you can try with --strand-filter 0 but i m not sure it will help.

ADD REPLYlink modified 15 months ago • written 15 months ago by Titus810

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?

ADD REPLYlink written 15 months ago by cschu1811.6k
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