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This horse deader than dead when ILMN's lawyer team destroyed the community college kid who worked for free this week...
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@ 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?Comment
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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.Comment
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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.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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