454 Amplicons-no reference

[dina]

Hello,
I wanted to know whether there is some option to analyze amplicon run with many samples barcoded with MIDs if I only have the primers and not the reference? Is it possible with the 454 software? with any other software?
And generally, are there other good softwares for analyzing amplicons barcoded with MIDS?
Thank you

[robs]

Can you be more specific what you mean with "analyzing"?

You can split your sequences by MIDS using the 454 software (command line program is called sffile). The MIDS are usually stored in the sff files and therefore, they are not required as input for sffile.

[dina]

1.how exactly to use sffile to split the samples?
2.I meant that I have very variable amplicons, when I enter a reference, most samples are not represented in the variations,,,,
Thank you a lot!

[robs]

1. Take a look at the 454 manuals (SW-Manual_PartC_Assembler-Mapper-SFFTools; section 3). If you want to convert the sff to e.g. fasta you want to use sffinfo.

2. Not sure what you mean with "when I enter a reference, most samples are not represented in the variations". Sorry.

[dina]

I meant that I use the AVA software, enter a reference and primers and planning the multiplexer. I have 30 samples. Then I receive the variations, but only in 2 samples. I thought it was because they are too variable??