Length and GC-biases during sequencing library amplification

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High throughput sequencing has broadened our understanding of countless biological processes and led to scientific advancements in  clinical therapeutics.   With massively parallel sequencing becoming a staple in laboratories worldwide, we need to understand the  limitations and biases of the technology. PCR amplification is an essential step in the preparation of a library for high throughput  sequencing.  However, biases introduced by PCR, reflecting the GC content and length of the template, can have detrimental effects on library generation. 

Dabney and Meyer have now compared ten commercially available PCR polymerase-buffer systems (Dabney and Meyer, Biotechniques 52:87-94, 2012) to determine the biases they introduce in sequencing both modern and ancient DNA.  For modern DNA library  preparation, Herculase II Fusion polymerase best maintained the GC content and length distribution of the library throughout 40 cycles of PCR, while Phusion polymerase in HF buffer introduced dramatic bias in both parameters relative to the original library.  Neanderthal DNA is a limited resource and contains a high level of GC-rich microbial contamination.  Of the ten polymerase-buffer systems tested for ancient DNA library preparation, AccuPrime Pfx produced the highest levels of endogenous sequences, while Phusion in HF buffer  preferentially amplifies the GC-rich microbial templates. 

PCR polymerases are a principal source of GC content and length bias in high throughput library preparation. Other uninvestigated  biases, such as thermocycling parameters, likely exist but optimizing the polymerase-buffer system can contribute to generating a library that accurately represents the starting material.

Desiree Boltz

  • Marlene Schooler

    Just read a really plausible novel called The Prophesy Gene. The main characters uncover a number of unintended genetic mutations as a result of the 1980s Aral Sea environmental disaster in Central Asia and the accidental release of a genetically modified strain of anthrax.  The author makes a pretty scary claim that mankind is stifling its own evolution by premeditated and accidental genetic engineering and mutations because we can’t possibly understand all of the consequences to ecosystems and dormant genetic sites and the food chain when we monkey with this stuff.  For example, some people eat oxen that have grazed on mutated vegetation and those people’s digestive systems irreparably stop working.  Or some dangerous fungus that humans eradicate because it causes disease but they don’t realize that it also sequesters carbon dioxide and could reverse global warming.  But I think the best one is that if it wasn’t for scientist’s genetic meddling, humans might one day evolve senses that bats and sharks have like hunting by their internal sonar or the ability that butterflies and some birds have to navigate by the earth’s magnetic field.  The book is by Stuart Schooler.  His website is http://www.stuartschooler.com and there’s a link to a blog and a Youtube video (http://vimeo.com/53365895).