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  • Tophat strand-specific RNAseq- Do i need to reverse-complement?

    Hi all
    I have strand-specific RNAseq data ( using Truseq kit- dUTP method) and I aligned the reads onto the genome with Tophat using --library-type=fr-firststrand option.
    But when I visualized the output ( in Artemis), it was not strand-specific. But then with a weird guess, I reverse-complemented the fastq file of Read1 and voila- there was strand-specificity. Could anyone explain this to me. I could not find any mention of this tweak anywhere (including the latest nature methods paper). Is it something very natural to do that no one mentions it?? Im a novice in bioinformatics analysis, so not able to grasp this concept!
    Thanks in advance!

  • #2
    Dear Abhinay,

    I used the same fr-firststrand with tophat but didn't see the strand specificity.

    Could you please share more in detail about the reverse complimenting step that you did?

    Thanks in advance.

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