mothur in 454 2.8 software

Hi there,
we are running mothur with sequences generated in the 2.8 FLX+ software version and we found that non of the sequences pass the filtering. there are several things changing in this new version:
1.- the flows are random, there is no sequential and repetitive flow, this may affect for calculating the flowgram and for that reason not getting sequences pass through?
2.- the number of flows now is 1812, can this affect to hte program functionality?

Other changes can be seen in the sequencing that may affect to the performance of mothur with 454 FLX + 2.8.

Can you please tell me if you have in mind changes for adapting mothur to this new version of FLX sequencing?

I will appreciate an answer.

I can send some files for you to see how this is now built.

All the best
PACO

1.- the flows are random, there is no sequential and repetitive flow, this may affect for calculating the flowgram and for that reason not getting sequences pass through?

Random? I’d like to see that. Can you send us one of your sff files?

2.- the number of flows now is 1812, can this affect to hte program functionality?

Shouldn’t be a problem. But you would need to re-sequence your own mock community to figure out an appropriate number of min and max flows.

Btw, congrats - you might be the only person to get data out of the new platform.
Pat

Hi there,
We do have the 2.8 sofware since september this year.
Please let me know how can I provide you the sff,
Can you provide an email, can you send me an email?
All the best
PACO

can you email a part of it to mothur.bugs@gmail.com?

Thanks for sending the sff file…

1.- the flows are random, there is no sequential and repetitive flow, this may affect for calculating the flowgram and for that reason not getting sequences pass through?

The flows aren’t random - if you run sffinfo(sff=454_reads_AC_A.MID10.sff, sfftxt=T) and look at the “Flow Chars” line, the flow order is TACG - this is typical.

2.- the number of flows now is 1812, can this affect to hte program functionality?

This shouldn’t affect the program. However, it will affect how you handle errors. We always recommend using a mock community when sequencing to assess the error rates. We find that the number of good flows is significantly lower than the number of total flows. For example, with GS FLX Titanium we are only using 450 flows of the 800 total. This was described in our PLoS ONE paper. You would need to do the same with this new platform.

Pat

Dear Pat,
If you compare the flow file your program create with the one I generated with the sfffile of 2.8 version that I sent to you, you will see that they do not match

PACO

Yeah it looks like the flow data are in a different format. I’m not sure what the question is since the sff file is in the same format as before and that is the input to sffinfo to start the SOP.