Two suggestions:

1. Change your strings

2. Listen to the tuning *unplugged*. It might be a physical problem.

Idea: (You may already know this, but I will post it anyway for the benefit of the forum)

Tuning is a funny thing. For example, the note E needs to be tuned higher as the 5th of an A chord (A-C#-E) than as the 3rd of a C chord (C-E-G) to blend just right. Singers, violin players, trombone players, etc. do this instinctively. But on instruments like piano and guitar, where a note can only have one pitch, each note is tuned to the average spot so that it comes close, but is not perfect, for all chords.

If you tune so that the G string sounds just right on the 1st fret in the E chord (G# as the 3rd, E-G#-B) then it will sound flat open in a C chord (as the 5th, C-E-G). The only solution is to compromise - make the G string a little flat for the C chord but a little sharp for the E chord.

This "compromise" system is called equal temperament and is what electronic tuners automatically use.

probably  the  only thing  thats  gonna  fit  under  the   strings  is an old   top mount  guitar  pickup or if you know  someone  with a  dogear p90 laying  around or someone  with   a roland  gk pickup or  maybe  one of those  thin  acoustic  pickups. what  you could  try  is  getting  a  support  ticket that  would   put  you  intouch  with   a line 6 tech.  i dont  know  how  to do that  because  ive  never  had  to but    i know   that  you can   work  one on one  with  a tech  that way.

  the  trouble  woth  the  usb  audio  interface  is  getting  it  connected  to the  guitar if you look at the  attatched  photo  of  thw  workbench  hardware   theres  two cables    and  the  interface. so  it  changes  the  guitar connector  to  usb  that way. i think  theres  a  chip  in there  that does  something  too.

   yeah  there is  i just looked  at it  so it  probably  has  some  miniscule  program  on it.

Hello,

   Outside of the software reflash and changing strings (to make sure there is not a string issue), you may want to have a qualified guitar luthier/tech go through the guitar to make sure there are no other problems with the nut or saddle piece that could be causing some other issue that would knock the intonation out of tune.  You can also take a look at this Peterson FAQ where they discuss how to correctly intonate and setup a guitar, as well as the ideas behind tempered tunings:

  http://www.petersontuners.com/index.cfm?category=85

   Please be sure to award Helpful and/or Correct points to community members who respond with helpful or correct answers, and close out the ticket as “answered” if your situation has been resolved.

Hello,

    It's been a couple of days since we've heard from you. We're going to assume your question has been answered and close this thread. If you still have questions please feel free to open a new question with us and we will answer your questions accordingly. Thanks!