Hi,

I do not know if anyone has asked the similar question or has met the similar problem.

I have an RNA-seq (from Illumina, 75 bp) data. When I am aligning the reads with bwa to zebrafish genome and count the number of reads with featureCounts I got the following summary:

                            Sample-1
Assigned                    9265562
Unassigned_Ambiguity        147537
Unassigned_MultiMapping     0
Unassigned_NoFeatures       3884814
Unassigned_Unmapped         265481
Unassigned_MappingQuality   0
Unassigned_FragmentLength   0
Unassigned_Chimera          0
Unassigned_Secondary        0
Unassigned_Nonjunction      0
Unassigned_Duplicate        0

When I am aligning with topHat2 and count the number of reads with featureCounts I got the following summary:

Status    accepted_hits.bam
Assigned    197566
Unassigned_Ambiguity    3850
Unassigned_MultiMapping    114795
Unassigned_NoFeatures    54359
Unassigned_Unmapped    0
Unassigned_MappingQuality    0
Unassigned_FragmentLength    0
Unassigned_Chimera    0
Unassigned_Secondary    0
Unassigned_Nonjunction    0
Unassigned_Duplicate    0

The alignment summary in align_summary.txt is:

Reads:
          Input     :    344347
           Mapped   :    278609 (80.9% of input)
            of these:     22834 ( 8.2%) have multiple alignments (2 have >20)
80.9% overall read mapping rate.

Why do I have such a big difference between the alignments? Moreover, the topHat alignment also has the following summary in bowtie.left_kept_reads.log:

16839634 reads; of these:
  16839634 (100.00%) were unpaired; of these:
    5058477 (30.04%) aligned 0 times
    10330251 (61.34%) aligned exactly 1 time
    1450906 (8.62%) aligned >1 times
69.96% overall alignment rate

Thanks in advance

ilyas