bam-readcount fails to report on all alignments in bam file

Question

[mark.rose]

Hi All

I’m using bam-readcount on a bam file with ~60000 alignments (stitched reads which span the 374 bp length of the amplicon reference).

bam-readcount   -w -1 -q 0 -b 0 -f my_ref.fa  sort.bam > bam_readcount.q0b0_w-1  2> bam_readcount.error

Here is a “head” of the output:

my_ref  1       G       7999    =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00        G:7999:44.00:37.95:44.00:7999:0:0.00:0.00:0.00:0:0.00:0.00:0.00 T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
my_ref  2       A       8000    =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:8000:44.00:37.83:44.00:8000:0:0.00:0.00:0.00:0:0.00:0.00:0.00 C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00        G:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
my_ref  3       T       8001    =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00        G:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  T:8001:44.00:37.96:44.00:8001:0:0.00:0.00:0.00:0:0.00:0.00:0.00 N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
my_ref  4       G       8002    =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:1:44.00:11.00:44.00:1:0:0.00:0.00:0.00:0:0.00:0.00:0.00       C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00        G:8000:44.00:37.94:44.00:8000:0:0.00:0.00:0.00:0:0.00:0.00:0.00 T:1:44.00:38.00:44.00:1:0:0.00:0.00:0.00:0:0.00:0.00:0.00       N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00

Note that only about 8000 reads are reported for each position and this is true for each position in the reference. Why are not all 60000 reads accounted for? Interestingly I get warnings for 8020 reads (about the number of alignments reported in the output)

WARNING: In read M00645:234:000000000-AT4W2:1:1106:7492:11191: Couldn't find the generated tag.

This is repeated 374 times for each read with a warning.

Thanks for your help

Mark