TopHat+BWT = reads that failed to align ~ 99%

[repinementer]

I ran Tophat and cufflinks successfully!
But I think some the output results went wrong (red colour ) but I'm not sure.

I ran 17246957 reads and my TopHat outputs are
accepted_hits.sam = 152434
junctions.bed = 12672

logs
$ cat file2lqYQ1.log

# reads processed: 17184877
# reads with at least one reported alignment: 17194 (0.10%)
[COLOR="Red"][B]# reads that failed to align: 17156835 (99.84%)[/B][/COLOR]
# reads with alignments suppressed due to -m: 10848 (0.06%)
Reported 127509 alignments to 1 output stream(s)

$ cat fileIw0Tgh.log

# reads processed: 17184877
# reads with at least one reported alignment: 69661 (0.41%)
[COLOR="Red"][B]# reads that failed to align: 17098690 (99.50%)[/B][/COLOR]
# reads with alignments suppressed due to -m: 16526 (0.10%)
Reported 487470 alignments to 1 output stream(s)

$ cat prep_reads.log

prep_reads v1.0.13
---------------------------
62080 out of 17246957 reads have been filtered out

segment_juncs v1.0.13
---------------------------
Loading reference sequences...
        Loading chr1...done
        Loading chr2...done
        Loading chr3...done
        Loading chr4...done
        Loading chr5...done
        Loading chr6...done
        Loading chr7...done
        Loading chr8...done
        Loading chr9...done
        Loading chr10...done
        Loading chr11...done
        Loading chr12...done
        Loading chr13...done
        Loading chr14...done
        Loading chr15...done
        Loading chr16...done
        Loading chr17...done
        Loading chr18...done
        Loading chr19...done
        Loading chr20...done
        Loading chr21...done
        Loading chr22...done
        Loading chrX...done
        Loading chrY...done
        Loading chrM...done
Found 0 potential split-segment junctions
Indexing extensions in Tophat_Brain/tmp//left_kept_reads_missing.fq
Total extensions: 394607205
Looking for junctions by island end pairings
Adding hits from segment file 0 to coverage map
Map covers 382631 bases
Map covers 374273 bases in sufficiently long segments
Map contains 8351 good islands
417440 are left looking bases
417332 are right looking bases
Collecting potential splice sites in islands
reporting synthetic splice junctions...
Examining donor-acceptor pairings in chr20
Examining donor-acceptor pairings in chr21
Examining donor-acceptor pairings in chr22
Examining donor-acceptor pairings in chr19
Examining donor-acceptor pairings in chr18
Examining donor-acceptor pairings in chr11
Examining donor-acceptor pairings in chr10
Examining donor-acceptor pairings in chr13
Examining donor-acceptor pairings in chr12
Examining donor-acceptor pairings in chr15
Examining donor-acceptor pairings in chr14
Examining donor-acceptor pairings in chr17
Examining donor-acceptor pairings in chr16
Examining donor-acceptor pairings in chrX
Examining donor-acceptor pairings in chrY
Examining donor-acceptor pairings in chr2
Examining donor-acceptor pairings in chr3
Examining donor-acceptor pairings in chr1
Examining donor-acceptor pairings in chr6
Examining donor-acceptor pairings in chr7
Examining donor-acceptor pairings in chr4
Examining donor-acceptor pairings in chr5
Examining donor-acceptor pairings in chr8
Examining donor-acceptor pairings in chr9
Found 865 potential island-end pairing junctions
done
Looking for junctions between and within islands
Adding hits from segment file 0 to coverage map
Recording coverage islands
Found 62807 islands covering 2120609 bases
Collecting potential splice sites in islands
reporting synthetic splice junctions...
Examining donor-acceptor pairings in chr20
Examining donor-acceptor pairings in chr21
Examining donor-acceptor pairings in chr22
Examining donor-acceptor pairings in chr19
Examining donor-acceptor pairings in chr18
Examining donor-acceptor pairings in chr11
Examining donor-acceptor pairings in chr10
Examining donor-acceptor pairings in chr13
Examining donor-acceptor pairings in chr12
Examining donor-acceptor pairings in chr15
Examining donor-acceptor pairings in chr14
Examining donor-acceptor pairings in chr17
Examining donor-acceptor pairings in chr16
Examining donor-acceptor pairings in chrX
Examining donor-acceptor pairings in chrY
Examining donor-acceptor pairings in chr2
Examining donor-acceptor pairings in chr3
Examining donor-acceptor pairings in chr1
Examining donor-acceptor pairings in chr6
Examining donor-acceptor pairings in chr7
Examining donor-acceptor pairings in chr4
Examining donor-acceptor pairings in chr5
Examining donor-acceptor pairings in chr8
Examining donor-acceptor pairings in chr9
Found 173876 potential intra-island junctions
done
Reporting potential splice junctions...done
Reported 174467 total possible splices

[frozenlyse]

What's your mapping rate if you just run them through bowtie instead of tophat?

[repinementer]

it is the same just with bowtie

why ~90% are failed ?
I just used Eric Wang's public data means it is good data.

[DrD2009]

Did you remember to clip the adapters?

From experience I once aligned a Fastq file containing adapters and received a very similar low percentage alignment.

That's my guess.

[moritz]

Hey repinementer,

did you solve this problem by clipping adapters and do you know the actual meaning of the different log files you posted? I get similar log files with different postfixes (fileXYZ.log) that contain different entries and I cannot make much sense out of them.

Besides, does someone know if it is appropriate to just divide the number of lines in the accepted_hits.sam file from TopHat by the total number of reads in order to get the overall ratio of aligned reads?

Best Moritz

[northbio]

hi,drd2009,
could you tell me how to clip the adapters?

[skycreative]

hi,drd2009,
could you tell me how to clip the adapters?

suggest you trying to find out the pipeline for seq-deal
It is not difficult to get the "clean" sequence