samstat versus tophat overall alignment rate %

[alyamahmoud]

Hi group

I am trying to map RNAseq single straneded paired end reads using tophat, the alignment rate I get using samtools falgstat is very good (~90%) however checking the logs files of tophat, specifically, bowties.left.keep.reads shows a very low overall alignment rate (~36%). Can someone please help me understand these parameters ?

Thanks a lot
Alyaa

This is the tophat command I use:

$ tophat -g 1 -p 8 -G test.gtf -o test_thout --library-type fr-firststrand test R1_001.fastq R2_001.fastq

This is the samtools flagstast command:

 $ samtools flagstat accepted_hits.bam 
36919320 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
36919320 + 0 mapped (100.00%:-nan%)
36919320 + 0 paired in sequencing
18510302 + 0 read1
18409018 + 0 read2
33270470 + 0 properly paired (90.12%:-nan%)
35338762 + 0 with itself and mate mapped
1580558 + 0 singletons (4.28%:-nan%)
406722 + 0 with mate mapped to a different chr
87168 + 0 with mate mapped to a different chr (mapQ>=5)

The logs files; bowtie.left_kept_reads.log

19786770 reads; of these:
  19786770 (100.00%) were unpaired; of these:
    12585633 (63.61%) aligned 0 times
    6908036 (34.91%) aligned exactly 1 time
    293101 (1.48%) aligned >1 times
36.39% overall alignment rate

[dpryan]

The accepted.bam file only contains mapped reads, not all of them, so the metrics will never be the same.