All SAM alignments on reverse strand

[dhanson]

I just went through my first TopHat analysis, and am worried about the output. There are actually two things that concern me:

1. The bitwise FLAG in the SAM file is 16 for every read. I know that 16 means a read from the reverse strand. But shouldn't half of the reads map to the reverse, and the other half to the forward? I used TruSeq3 Stranded for my library prep. If my reads are only mapping to the reverse strand, doesn't that mean I will miss the expression of any gene that resides on the forward strand?

2. TopHat aligned 84.1% of reads (~29 million) to the genome, of which 7.9% (~2 million) were mapped multiple times. Since I chose the option allow a maximum of 1 alignment per read, I would expect my SAM file to be approximately 29 million lines long. However, it is about 32 million lines long! How can this be?

Thanks to anyone who can help me with this.

[fanli]

Can you post your tophat command?

1) What annotation files did you use and what options did you specify...maybe you have a weird edge case
2) again depends on options passed - did you use --max-multihits?

[dhanson]

I'm actually using Galaxy to run TopHat. I left all the settings as default, except that the option "Maximum Number of Alignments to be Allowed" I set to 1. For the annotation file I used the whole genome of my species (Gasterosteus aculeatus) in fasta format. There is no annotation in this file (I was going to use the annotation file later when using Cufflinks).

For the first issue (every hit is on the reverse strand), I thought it might have something to do with the fact that I used stranded library prep? For "Library Type" option in tophat, I left it as "FR unstranded" because I thought that the other two options were for paired-end reads (I have single-end).

Thanks for your input and help!

[Bukowski]

Strandedness has to do with the library preparation, not whether you've done single or paired-end sequencing.

[dhanson]

Thanks. But if I am to select one of the stranded options in tophat, how do I know whether to choose forward or reverse strand for the "Library Type" option?

[Bukowski]

You ask your sequencing provider how they prepared the library..

And use this as a guide: https://www.biostars.org/p/64250/

Edit: I see you used TruSeq stranded so fr-firststrand is appropriate

[dhanson]

I just finished another TopHat run this time with "Library Type" set to FR-firststrand, and got the exact same results. Every FLAG is 16, and the SAM is much longer than the reported number of aligned reads.

Any more possible settings that could be causing this?

Thanks again for your input