How to read my Samtools mpileup?

Question

[c.clarido]

Hello community,

I have entered the following command:

 time samtools mpileup -f /home/bnexta mapped.sorted.bam > variants.mpileup

And If i read the head of the file, I get the following:

11567   892 t   1   ^?. G
11567   893 t   1   .   G
11567   894 t   1   .   G
11567   895 t   1   .   G
11567   896 g   1   .   G
11567   897 a   1   .   G
11567   898 a   1   .   G
11567   899 g   1   .   G
11567   900 a   1   .   G
11567   901 t   1   .   G

According to http://samtools.sourceforge.net/pileup.shtml , the default output should look like this:

seq1 272 T 24  ,.$.....,,.,.,...,,,.,..^+. <<<+;<<<<<<<<<<<=<;<;7<&
seq1 273 T 23  ,.....,,.,.,...,,,.,..A <<<;<<<<<<<<<3<=<<<;<<+
seq1 274 T 23  ,.$....,,.,.,...,,,.,...    7<7;<;<<<<<<<<<=<;<;<<6
seq1 275 A 23  ,$....,,.,.,...,,,.,...^l.  <+;9*<<<<<<<<<=<<:;<<<<
seq1 276 G 22  ...T,,.,.,...,,,.,....  33;+<<7=7<<7<&<<1;<<6<
seq1 277 T 22  ....,,.,.,.C.,,,.,..G.  +7<;<<<<<<<&<=<<:;<<&<
seq1 278 G 23  ....,,.,.,...,,,.,....^k.   %38*<<;<7<<7<=<<<;<<<<<
seq1 279 C 23  A..T,,.,.,...,,,.,..... ;75&<<<<<<<<<=<<<9<<:<<

Obviously something is off. According the link provided above, the columns from left left to rightshould be the chromosome, reference base, number of reads covering the site, readbases, base qualities. So If I take the second line of my result: chomosome: 893, reference base: t, counts: 1, readbase: . and quality: G

The count, readbase and quality seems off to me. I hope someone explain to me what seems to be wrong with the output?

*Note that in my pipeline I used 245713 reads to test, originally I have 12,819,328 reads.

Answer 1

[finswimmer]

Hello,

the first column is the chromosome name, the second the 1-based position. It looks like the sites you are showing are just covered by 1 read. Use igv or something similar to take a visual look on this position and check if this is correct.

fin swimmer