Paired-end sequencing for functional genes?

[chwang]

I will be trying to sequence a specific set of functional genes with the 454 GSJunior...Their size, for example, can range from 800-900 bp. I was wondering if using paired-end sequencing would be the approach to get full sequence length for the respective genes? The idea is to load multiple functional genes onto one lane, each with respective MIDs. I was confused since I thought paired-end was mainly for whole genome sequencing? If I were to use paired-end sequencing, how is the primer design different than 16S rRNA amplicon sequencing? Thanks!

[ajthomas]

No, you don't want to do paired-end sequencing for this. If I understand right, you will be amplifying these genes and then sequencing the amplicons, right? If so, just use the Lib-A adapters, rather than Lib-L, and you will get sequences from both ends. You won't sequence any individual bead from both ends, as you would with Illumina sequencing, but you will sequence some molecules from one end and others from the other end. After sequencing you will assemble the forward and reverse reads into full length sequences.

[chwang]

Hi. Thanks for the response. Could you explain how it would be possible to determine which forward and reverse sequences to stitch together? ...How to determine that both ends were sequence from the same DNA molecule? Because is I'm using the functional gene primers onto an environmental sample, not bacterial isolates.