TopHat alignment issue

[telos]

Hello,

I have encountered a situation where error-free reads generated from a cDNA fasta file do not align to the correct transcripts. Instead, they align to the junction of a different transcript. Does anyone have any idea why this is? Is it due to a bug?

Below is a series of 23 reads to demonstrate the problem (and below that the SAM output). The IDs of the reads (before the '.' character) correspond to the true transcripts which produced them (the sequences perfectly match the Ensembl v56-annotated reference). If you run TopHat you will find that the reads align to a completely incorrect transcript --- the junction of ENSMUST00000111033 --- and not to the correct transcripts.

I used option -G with a GFF file derived from the Ensembl v56 mouse GTF file.

Many thanks
Ernest.

>ENSMUST00000034431.1
TTGTTTGTTTGTTTGTTGTTGTTTTTGTTTTGTTT
>ENSMUST00000031124.46
TGTTTGTTTGTTTGTTTTTTGTTTTGTTTTGTTTT
>ENSMUST00000116594.499
TGTTTGTTTTTTGTTTTGTTTTGTTTTGTTTTGTT
>ENSMUST00000119430.145
TCTTTGTTTTTGTTTTTGTTTTATTTTGTTTTGTT
>ENSMUST00000090791.46
TTTTTTTTTTTTTGTTTTGTTTTGTTTTGTTTTGT
>ENSMUST00000034136.73
TTTTTGTTTTTGTTTTGTTTTGTTTTGTTCTTTTT
>ENSMUST00000086538.200
TTTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTT
>ENSMUST00000002145.103
AAAACAAAACAAAACAAAACAAAACAAAACAAAAA
>ENSMUST00000086909.75
AAAACAAAACAAAACAAAACAAAACAAAACCAACA
>ENSMUST00000115059.42
AAAACAAAACAAAACAAAACAAAACAAAAAAAAAA
>ENSMUST00000002145.140
CAAAACAAAACAAAACAAAACAAAACAAAACAAAA
>ENSMUST00000002145.33
CAAAACAAAACAAAACAAAACAAAACAAAACAAAA
>ENSMUST00000023147.258
CAAAACAAAACAAAACAAAACAAAACAAAACCAAA
>ENSMUST00000086909.105
CAAAACAAAACAAAACAAAACAAAACAAAACCAAC
>ENSMUST00000086909.80
CAAAACAAAACAAAACAAAACAAAACAAAACAAAA
>ENSMUST00000002145.15
ACAAAACAAAACAAAACAAAACAAAACAAAACAAA
>ENSMUST00000002145.124
AACAAAACAAAACAAAACAAAACAAAACAAAACAA
>ENSMUST00000002145.142
AACAAAACAAAACAAAACAAAACAAAACAAAACAA
>ENSMUST00000078755.138
AACAAAACAAAACAAAACAAAACAAAACAAAAAAA
>ENSMUST00000078755.58
AACAAAACAAAACAAAACAAAACAAAAAAACAACA
>ENSMUST00000098666.32
AACAAAACAAAACAAAACAAAACAAAACAAAACAA
>ENSMUST00000098668.78
AACAAAACAAAACAAAACAAAACAAAACAAAACAA
>ENSMUST00000098672.52
AACAAAACAAAACAAAACAAAACAAAACAAAACAA

@HD VN:1.0 SO:sorted
@PG TopHat VN:1.0.13 CL:/home/et04/bin/tophat -G /work/et04/GFFfiles/Mus_musculus.NCBIM37.56.adapted.gff -o littletest3.results -p 8 mm9 /work/et04/RNAseqData/simulatedmouse/littletest.fasta
ENSMUST00000034431.1 0 chr2 109766876 255 23M179N12M * 0 0 TTGTTTGTTTGTTTGTTGTTGTTTTTGTTTTGTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:1 XS:A:+ NS:i:1
ENSMUST00000031124.46 0 chr2 109766877 255 22M179N13M * 0 0 TGTTTGTTTGTTTGTTTTTTGTTTTGTTTTGTTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000116594.499 0 chr2 109766885 255 14M179N21M * 0 0 TGTTTGTTTTTTGTTTTGTTTTGTTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000119430.145 0 chr2 109766885 255 14M179N21M * 0 0 TCTTTGTTTTTGTTTTTGTTTTATTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000090791.46 0 chr2 109766889 255 10M179N25M * 0 0 TTTTTTTTTTTTTGTTTTGTTTTGTTTTGTTTTGT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000034136.73 0 chr2 109766891 255 8M179N27M * 0 0 TTTTTGTTTTTGTTTTGTTTTGTTTTGTTCTTTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000086538.200 0 chr2 109766891 255 8M179N27M * 0 0 TTTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000002145.103 16 chr2 109766892 255 7M179N28M * 0 0 TTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000086909.75 16 chr2 109766892 255 7M179N28M * 0 0 TGTTGGTTTTGTTTTGTTTTGTTTTGTTTTGTTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000115059.42 16 chr2 109766892 255 7M179N28M * 0 0 TTTTTTTTTTGTTTTGTTTTGTTTTGTTTTGTTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:1 XS:A:+ NS:i:1
ENSMUST00000002145.140 16 chr2 109766893 255 6M179N29M * 0 0 TTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000002145.33 16 chr2 109766893 255 6M179N29M * 0 0 TTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000023147.258 16 chr2 109766893 255 6M179N29M * 0 0 TTTGGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:1 XS:A:+ NS:i:1
ENSMUST00000086909.105 16 chr2 109766893 255 6M179N29M * 0 0 GTTGGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000086909.80 16 chr2 109766893 255 6M179N29M * 0 0 TTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000002145.15 16 chr2 109766894 255 5M179N30M * 0 0 TTTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000002145.124 16 chr2 109766895 255 4M179N31M * 0 0 TTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000002145.142 16 chr2 109766895 255 4M179N31M * 0 0 TTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000078755.138 16 chr2 109766895 255 4M179N31M * 0 0 TTTTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:1 XS:A:+ NS:i:1
ENSMUST00000078755.58 16 chr2 109766895 255 4M179N31M * 0 0 TGTTGTTTTTTTGTTTTGTTTTGTTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000098666.32 16 chr2 109766895 255 4M179N31M * 0 0 TTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000098668.78 16 chr2 109766895 255 4M179N31M * 0 0 TTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2
ENSMUST00000098672.52 16 chr2 109766895 255 4M179N31M * 0 0 TTGTTTTGTTTTGTTTTGTTTTGTTTTGTTTTGTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 XS:A:+ NS:i:2