The Meaning Of "Seq Being Reverse Complemented" In Sam Format

Question

[Pascal]

Hi

After reading the SAM specs it is still not very clear what is the meaning of these two fields in the FLAG:

• SEQ being reverse complemented
• SEQ of the next segment in the template being reversed

Could you please explain it to me?

UPDATE: if I understood it well when "SEQ being reverse complemented" is NOT SET it means that the read has been aligned to the forward strand, when SET it means that it has been aligned on the reverse strand. Is that correct?

And can we use these fields to detect an incorrect orientation of a paired-end read ?

Regards, Pascal

Answer 1

[Istvan Albert]

As you mention it means that the reverse complement of the read aligns to the position that is indicated; and that is equivalent to saying that it aligns to the complementary strand in reverse direction.

The reason for the wording is probably to remind you that the position field is still the leftmost position of the alignment and not the actual 5' start of the read.

You may use this information to detect properly paired reads, although there is more to these than just being in the correct orientation, for example the distance between them and other considerations may need to be taken into account. Flag 2, read mapped in proper pair should be filled in by the aligner.