Interpreting Flags in SAM file

Question

[jayg]

Hi,

I have Illumina pair ended reads mapped to contigs. All the pair ended reads have innie (------> <----------) orientation. I want to find orientation of contigs inferred from reads. Please see following cases

1. When the read is first in pair and is mapped to forward strand:

----------------------------------> contig

------------> read

1. When the read is first in pair and mapped to reverse strand:

<---------------------------- contig (rev complemented)

-----------> read

1. When the read is second in pair and mapped to forward strand:

<---------------------------- contig (rev complemented)

<------------

1. When the read is second in pair and mapped to reverse strand:

------------------------------> contig

<------------ read

Is the correct way to interpret?

Thank you very much.

[Istvan Albert]

It is not entirely clear what you are asking:

samtools flags

will tell you all the flags that you can use. Then you can add that to the -F and/or -f as needed (you can combine into a single number but sometimes is more explicit to list each separately). For example reads that are: mapped, first in pair, forward strand

-F 4 -f 64 -F 16

Answer 1

[Pierre Lindenbaum]

you're wrong with first and second in pair: first and second means that if the read comes from a paired-end experiment (output was something like R1.fastq.gz and R2.fastq.gz), then "1st in pair" was a read from R1.fastq.gz and "2nd in pair" came from "R2.fastq.gz". And that's it, there is no indication about the position.

Answer 2

[John]

Basically, it all comes down to if the sequencing was stranded - i.e. during PCR the original strand of DNA being amplified is some how recorded.

Maybe this post will help you: How many ways a read pair can be mapped?