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Samtools fields explained !: Revision

From the SAM format specification, the fields are:

Column Field Type Regexp/Range Brief description
1 QNAME String [!-?A-~]{1,255} Query template NAME
2 FLAG Int [0,216-1] bitwise FLAG
3 RNAME String \*|[!-()+-<>-~][!-~]* Reference sequence NAME
4 POS Int [0,231-1] 1-based leftmost mapping POSition
5 MAPQ Int [0,28-1] MAPping Quality
6 CIGAR String \*|([0-9]+[MIDNSHPX=])+ CIGAR string
7 RNEXT String \*|=|[!-()+-<>-~][!-~]* Ref. name of the mate/next read
8 PNEXT Int [0,231-1] Position of the mate/next read
9 TLEN Int [-231+1,231-1] observed Template LENgth
10 SEQ String \*|[A-Za-z=.]+ segment SEQuence
11 QUAL String [!-~]+ ASCII of Phred-scaled base QUALity+33

The two bitwise flags for the reads are 99 and 147, which indicate:

99 - read paired, read mapped in proper pair, mate reverse strand, and first in pair
147 - read paired, read mapped in proper pair, read reverse strand, and second in pair

The RNEXT field is "=" because the mate-pairs mapped to the same reference. The PNEXT field shows the position of the mate pair, so for r1 this shows the position of r2 and vice versa. The TLEN is +/- 400, which is equal to the insert size of the fragment created.