WGA question

[joskee]

Dear all,

I have a question about uneven amplification during WGA.
I received a sheet with the results of a sequence analysis, however I am not an expert in this field and wonder what they mean by this.
They stated that WGA may cause uneven amplification, leaving some regions extremely abundant in sequencing libraries while others have little or no representation, it is one possible reason why there is relative high rate of unique and low-frequency K-mer.

So not sure what they mean... Do they mean that certain parts of DNA are amplified more then others? If so: how come ? Isnt WGA a random proces?

Thanks in advance.

[mastal]

WGA question

So not sure what they mean... Do they mean that certain parts of DNA are amplified more then others? If so: how come ? Isnt WGA a random proces?

yes, some regions are amplified more than others.

the process is random in theory, in practice many factors, such as GC content, may affect the amplification.

[phillie]

I have a follow-up question: can WGA cause changes in the heterozygosity of the sample?
I can imagine that if some areas are more amplified than others you also change the level of heterozygosity ?

[imijay]

WGA can definitely be uneven. MALBAC is a new method developed to help reduce this uneveness. Also, I'm not sure what your libraries look like before sequencing but mine are 'broad' ranging in size from ~300-1000bp (i'm using rubicon's picoplex kit). During sequencing the smaller fragments will sequence more than the larger and the resulting data will reflect this bias- just to note.

[grosswesir]

It is well documented that multiple strand displacment WGA methods have a high allelic drop out rate (ADO) and affect the heterozygostiy of your samples. Some WGA kits have been compared in these papers from 2014 here:
http://www.ncbi.nlm.nih.gov/pubmed/25136831
http://www.ncbi.nlm.nih.gov/pubmed/25066732