Hi all,
I am hoping to perform whole exome sequencing on small bronchial biopsies at a depth of 400X.
I was told that I may not have enough starting DNA.
How much starting material is needed for that depth?
Also, mechanistically, why does increasing depth require more starting amount of DNA? When a region of DNA is ‘read’, do the adaptors not serve to re-read the sequence so not sure why you’d need more initial DNA?
Hope that makes sense?
Many thanks,
Lukas
Hi there,
For deep whole exome sequencing, you should consider using a decent amount of gDNA for the library prep upstream of the actual hybrid capture to generate highly complex whole genome libraries. We usually start with 50ng hmw gDNA as input for QIAseq FX, a library prep protocol employing enzymatic fragmentation. Using such libraries in whole exome hybrid capture workflows, it‘s easily possible to sequence at ~400x without getting elevated levels of duplicate reads which typically go up when (whole genome or whole exome) libraries have low complexities.
Best, Peter
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