orientation of 454's reads

[louis7781x]

Hi,

I want to know 454's reads has orientation?

I mean orientation of row data is 5'---------------------3' ?

Because I know data from illumina can't determine orientation.

[Andrew_Slatter]

Yes, read data is 5'-3'. Reads start with TCAG key which are the last 3' bases of the 454A primer.

[louis7781x]

Yes, read data is 5'-3'. Reads start with TCAG key which are the last 3' bases of the 454A primer.

Thanks!!!!!!

[TonyBrooks]

Surely all sequence reads are 5' to 3'? DNA cannot extend in the other direction.

Do you mean, "which strand are do the reads come from"? That all depends on how the library was created. If it's amplicon sequencing, you can choose the strand by designing the PCR to a particular strand. If it's standard rapid library then the strandness of the read cannot be determined - just like on the Illumina.

[louis7781x]

Surely all sequence reads are 5' to 3'? DNA cannot extend in the other direction.

Do you mean, "which strand are do the reads come from"? That all depends on how the library was created. If it's amplicon sequencing, you can choose the strand by designing the PCR to a particular strand. If it's standard rapid library then the strandness of the read cannot be determined - just like on the Illumina.

Hi,In my research,I have used 454 to detect reads that span fusion junction,but i don't know which is upstream gene and which is downstream gene.

If reads's orientation is 5' to 3',I can suggest that gene of 5' is upstream and gene of 3' is downstram.

So,you mean if the library was created rapidly,I can't determine which orientation is correct?

[Andrew_Slatter]

Its hard to say what can be concluded without looking at experimental design.
Have you mapped the reads to your expected target ?
Did you capture the fusion junction by PCR ? If so, you should be able to determine polarity from primer design as TonyBrooks points out above.
It all depends how the 454 adaptor sequences were appended to your target.

[louis7781x]

Its hard to say what can be concluded without looking at experimental design.
Have you mapped the reads to your expected target ?
Did you capture the fusion junction by PCR ? If so, you should be able to determine polarity from primer design as TonyBrooks points out above.
It all depends how the 454 adaptor sequences were appended to your target.

Hi,we don't capture the fusion junction yet,but it is prepared to do.

Thanks for your recommendation,I think I need to check the detail of being created library.