How can I get the read depth of my polymorphism?

Question

[apl00028]

With the aim to know the number of read depth that I have in each polymorphisms of my RNAseq data:

I have done an alignment using bowtie to alignm reads against a reference sequence:

bowtie2 -f -x scaffold_filt_PV014_OD_DB.fa PV002_cmv3.fa -S scaffold_nofilt_OD_PV002.sam

I change sam format to bam format:

samtools view -bS scaffold_nofilt_OD_PV002.sam > scaffold_nofilt_OD_PV002.bam

I removed unpaired reads: samtools view -F 0x04 -b scaffold_nofilt_OD_PV002.bam >scaffold_nofilt_OD_rm_PV002.bam

I select those with a map quality higher than 10

samtools view -q 10 -b scaffold_nofilt_OD_rm_PV002.bam > scaffold_nofilt_OD_MapQ10_PV002.bam

I sort and I index it:

samtools sort scaffold_nofilt_OD_MapQ10_PV002.bam scaffold_nofilt_OD_PV002_index
samtools index scaffold_nofilt_OD_PV002_index.bam

After to do this I get the coverage of each base pair using bedtools:

bedtools genomecov -ibam scaffold_nofilt_OD_PV002_index.bam -bga > coverage_PV002.txt

Moreover I got the number of polymorphisms using these functions:

samtools mpileup -g -f scaffold_filt_PV014_OD_DB.fa scaffold_nofilt_OD_PV002_index.bam > scaffold_nofilt_OD_PV002_index.bcf

bcftools view -v -c -g scaffold_nofilt_OD_PV002_index.bcf > scaffold_nofilt_OD_PV002_index.vcf

When I compare the read depth bedtools values against the bcftools values (of DP4 column) I got a big difference:

• With bcftools I get this type of read depth: 245,222,8,171,249,etc
• With bedtools I got read depths of 9248,9265,49971,50076.

I don't know what read depth value I should take

What is the difference?

Thanks in advantage.

[GenoMax]

I select those with a map quality higher than 10

Can you tell us how many reads did you lose after this step?

If you are purely looking to see how many reads cover a position then samtools depth -b <BED list of positions or regions> or mosdepth (https://github.com/brentp/mosdepth ) will work well.

[apl00028]

I don't remove any read with that function. But I will used samtools depth. Thanks in advantage.