understanding multi-mapped reads

Question

[Assa Yeroslaviz]

Hi everybody,

I am having a problem understanding the output of tophat concerning the multi-mapped reads. I would like to create a barplot of the unique vs. multi-mapped vs. unmapped reads from tophat-mapped bam files. (BTW is there already a tool for doing that?)

Now I wanted to extract the multi-mapped reads from one bam file. lat us say MW6. this is the align_summary.txt file I have:

Reads:
          Input     :  26140314
           Mapped   :  25159791 (96.2% of input)
            of these:   1027691 ( 4.1%) have multiple alignments (1832 have >20)
96.2% overall read mapping rate.

But when I try to count the reads manually I get this :

$samtools view bamFiles/MW6.sorted.bam | grep -w -v  "NH:i:1" | wc -l
4118642

and when I use the flagstat option from samtools:

$samtools flagstat bamFiles/MW6.SE.sorted.bam
28250742 + 0 in total (QC-passed reads + QC-failed reads)
3090951 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
28250742 + 0 mapped (100.00% : N/A)
0 + 0 paired in sequencing
... (the rest is 0)

The total number of input reads is 26140314 (as it says in the align_summary file). The total mapped reads from the bam file is 28250742, as says in the samtools flagstat. I guess this conflict is due to reads which mapped multiple times, but how can I get the exact number of multiple reads?

is it just 28250742 - 26140314 = 2110428 ?

Would it be similar in paired-end and single-end mapping runs?

thanks

Assa

Answer 1

[Carlo Yague]

The total mapped reads from the bam file is 28250742

This is the total number of alignments, which is different than the total number of reads. Reads can have 0, 1 or more alignments.

• There are 25159791-1027691=24132100 reads that map only 1 time.
• There are 1027691 reads that map multiple times.
• The 24132100 uni-reads generate 24132100 alignments.
• The 1027691 multi-reads generate 28250742-24132100=4118642 alignments including 3090951 secondary alignments.