weird sam flags

[capricy]

Hi, there,

I am trying to extract the mapped second read from a bam file (tophat output) using the following command:

====
samtools view -f 80 accepted_hits.bam|head -3

HWI-ST446:124987429:C0W8FACXX:6:1312:2288:18978 89 A_Cont1 3303 50 65M * 0 0 CACTGCTATATGGAGCGTACTGCCCATTATTCGCAATGCTACGATCAGCACAATCCAACCAATCGIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:65 YT:Z:UU NH:i:1
HWI-ST446:124987429:C0W8FACXX:6:2204:4776:94752 81 A_Cont1 3312 50 65M A_Cont103 386245 0 ATGGAGCGTACTGCCCATTATTCGCAATGCTACGATCAGCACAATCCAACCAATCGTCACCAAGA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:65 YT:Z:UU NH:i:1
HWI-ST446:124987429:C0W8FACXX:6:2108:2853:42840 89 A_Cont1 3542 50 59M1002N6M * 0 0 TCTCTGAGCACAGCCATCAGATCATCGATATCCTGAGAATAGCCATTCTGTCCAGGATTCTGCTC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XM:i:0 XO:i:0 XG:i:0 MD:Z:65 NM:i:0 XS:A:- NH:i:1
====

however all those flags, 89 or 81, indicate that they are actually the first read, according to the following website

https://broadinstitute.github.io/picard/explain-flags.html

Flag: 81

Explanation:
V read paired
read mapped in proper pair
read unmapped
mate unmapped
V read reverse strand
mate reverse strand
V first in pair
second in pair
not primary alignment
read fails platform/vendor quality checks
read is PCR or optical duplicate
supplementary alignment

anybody can help me figure out why?

Thank you very much!

[dpryan]

If you want the second read from a pair, then "-f 129" would make more sense.

[capricy]

Thank you for the message. It worked!