Hi, I have a couple questions regarding adapters+barcodes for paired-end mRNA-Seq. The way I understand it is that after mRNA fragmentation and first- and second- strand synthesis, you ligate the adapters, on either ends of the fragment. Here, I thought that the barcodes are on both ends of the fragment, between the adapter and the sequence... That is, in this fashion.
Adapter1, barcode, fragment, barcode, Adapter2
Basically, the procedure is then repeated two times, one reading from Adapter1 end and other from Adapter2 end. My understanding is that the barcode is the same.
My questions are:
1) Even if the barcodes are same, how does the sequencer know the matching pairs (or) how does it identify a particular pair come from the same fragment ?
2) Is it possible to sequence a paired-end read with barcodes only on one end? That is,
Adapter1, barcode, fragment, Adapter2.
How would this be possible/work?