Custom barcodes AND Paired end seq readlength

[GenomicIBK]

1. As the primersequences for the in-gel PCR in the RNA seq protocoll are known, has anyone ever tried it with custom primers (e.g. own barcode sequences)?

2. Paired end sequencing allows me to sequence 35 bp from the other direction (P2 adaptor). From which point are these 35 bp counted? Because when you use a barcode (+ internal adaptor) and these 35 bp are counted from the P2 adaptor, you lose a lot of bp just for these two (barcode and internatl adaptor) sequences.

Thank you!

[pmiguel]

(1) No, but we had an oligo company synthesize the primers designed on ABI bar codes--which are insanely expensive when purchased from ABI. Did not seem to be any issues with them.

(2) The bar code is determined in a 3rd read. It does not consume any of the sequence read space. So you can get full 75 nt forward reads and 35 nt reverse reads (of much lower quality, last I checked.) The 10 nt bar codes are collected in another read.

--
Phillip

[SOLiDance]

1) Can't use custom barcodes;cause you can only select barcode Num,but not input barcode sequence when you set up a seq run on the SOLiD machine~
2) agreed with phillip

[SeqAA]

If you can bin your own barcodes, you can make custom indexed adapters.

You would not be able to have the default software bin them for the reasons already stated.