Secondary Alignment in BAM flag

Question

[eudoraleer]

Hi all, was wondering what does it mean by secondary alignment in BAM flag?

What is been defined as unmapped reads?

Option used:

samtools view -F 260 bam_file

Thanks!

Answer 1

[Devon Ryan]

Unmapped reads are in the BAM file but have no valid assigned position (N.B., they may have an assigned position, but it should be ignored). It's typically the case that a number of reads can't be aligned, due to things like sequencing errors, imperfect matches between the DNA sequenced and the reference, random e. coli or other contamination, etc..

A secondary alignment occurs when a given read could align reasonably well to more than one place. One of the possible reported alignments is termed "primary" and the others will be marked as "secondary".

[eudoraleer]

Thank you Ryan, I have a small question, are secondary aligned reads considered duplicates?

[Devon Ryan]

I assume you mean PCR duplicates, to which the answer is no.

[eudoraleer]

Thanks, I think I am getting too confused with the terms duplicates, coverage and secondary reads, especially between duplicates and coverage, explanations in many posts does not seem to get me fully understood the meaning of pcr duplicates.

[giusdalt95]

I think they are... look at this post, maybe will clarify your doubt: How do I count the real total reads from a fastq file?

[Ram]

They are not. Pierre clearly mentions in your post (as did Devon here) that secondary alignments are reads that map at multiple positions, I don't understand why you had to hunt down a 6 year old post and add a wrong comment.