stranded coverage and reads alignment

[ysueha...@gmail.com]

Hi, 

I ran targeted sequence by Ion PGM. On the middle of amplicon, stranded coverage information showed that plus strand coverages are higher than those of minus (shown below), however it seems minus strand reads outnumber those of plus in colored alignment (image attached). 

 chr2:25,457,244

Total count: 35146

A : 73 (0%, 73+, 0- )
C : 28 (0%, 27+, 1- )
G : 35043 (100%, 32499+, 2544- )
T : 2 (0%, 2+, 0- )
N : 0
---------------
DEL: 0
INS: 255     

What could be wrong?

Thanks a lot, 

Yasu 

[MONA SPECTOR]

Hi, I am sorry to say I don't have an answer for this, but do have a related question as I am also wondering about this. How is the strand orientation determined for calculating the strand coverage? I have a preponderance of + reads although I expected 50%. Thanks for your help. Mona

[James Robinson]

Hi,

I'm actually not sure, its impossible to say from the screenshot.   I assume you are coloring by read strand.   Could you extract this portion of you bam and share it with us?   You can do this from the right-click menu.   I can send you a drop box link to upload.   

[James Robinson]

Read strand is determined from a flag in the BAM alignment record.