99.9% are discordant alignments, please help

Question

[dixit.anirudha]

Hi everyone,

I am using TopHat PE to align my RNA-sequences and in the alignment summary file I am finding this,

Left reads:
               Input:  27176120
              Mapped:  24040642 (88.5% of input)
            of these:    715407 ( 3.0%) have multiple alignments (0 have >20)
Right reads:
               Input:  27177333
              Mapped:  23904080 (88.0% of input)
            of these:    732099 ( 3.1%) have multiple alignments (0 have >20)
88.2% overall read alignment rate.

Aligned pairs:  22342472
     of these:     20162 ( 0.1%) have multiple alignments
          and:  22319633 (99.9%) are discordant alignments
 0.1% concordant pair alignment rate.

Not sure what is going on. On the other hand the if I use either R1 or R2 files in TopHat SE, the alignment score is 95%. Any inputs to resolve this will be great. Appreciated.

Answer 1

[Devon Ryan]

It appears that you trimmed the pairs separately, such that they're now out of sync. Search biostars for a large number of ways to resync the files (I think BBtools comes with a convenient tools for this).

[dixit.anirudha]

Thanks a ton Devon.