Query to call SNP in Illumina pair end sequencing

Question

[Bioinfonext]

Hi

I am trying to understand SNP calling in Illumina pair-end sequencing. I am having 30 genotypes od a species. After mapping individually all genotypes to reference with BWA and processing through samtools, After that we a bam file for each genotype.

Now I am not understanding mpileup step to call SNP for each genotype.

For given gene we can I have two alleles, so they may have some nucleotide difference within genotypes than How we can call them as SNP when comparing with reference. see position 7

           1 2 3 4 5 6 7 8 9

Refere:    A T A T A T G C G

for    1 : A T A T A T A C G

for    2 : A T A T A T A C G

for    3 : A T A T A T A C G

rev    4 : A T A T A T G C G

for pair-end sequencing for can be forward read and rev can be reverse read from opposite strand. I am confused.

Answer 1

[Brian Bushnell]

Don't worry about whether reads are paired or not, or from the forward or reverse strand. The aligner and variant-caller take care of all of that for you, and variants are reported according to the plus strand.

[Bioinfonext]

Thanks for reply. How genome sequence is assembled, if sometimes coding sequence present is opposite strand?

Like In Radish, there is 9 chromosome pair total 18 chromosomes. But in the genome sequence, only 9 chromosome sequence is considered as whole genome sequence.

I am taking those 9 chromosome sequences as the reference for SNP detection.

[Brian Bushnell]

Typically, if there are multiple copies of chromosomes, people only consider one of them, and consider SNPs with respect to the canonical copy.