[Elcannibal]

This horse deader than dead when ILMN's lawyer team destroyed the community college kid who worked for free this week...

[Dolphin22]

@ singlemoleculer
Thanks for your advices. I checked my sequences but I am not sure about some points:

What does very rich of A's and T's mean? Do you have a percentage? The same with the low number of G's.
Of course I find CTAG's but where is the limit?

I also find many sequences with long stretches of polyA's polyT's? Is this also an indicator for bad sequencing?

[singlemoleculer]

The standard Helicos filtering software gets rid of these artifacts automatically. If you don't have access to the software, you can do manual filtering. Very rich in AT means A+T>90%. These are usually caused by problems in sample preparation. Any such sequences should be discarded unless you are working with a very AT-rich set of sequences like Plasmodium in which case you have to adjust the cutoff. For CTAG, you should look at the dimer frequencies. If CT+TA+AG+GC>70%, you should also discard. These sequences are usually caused when 2 DNAs are too close on the flow cell and the signals overlap. The filtering software actually has a more complex algorithm but this is good enough for most purposes.

[Dolphin22]

Thanks for your reply.
I did use the Helipshere software for the analyis so the artifacts should be removed automatically. But I will check the nucleotide distribution of my unaligned sequences allos manually.