Optimized whole-genome amplification strategy for extremely AT-biased template.
Bottom Line: We present a strategy for whole-genome amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing.We show that this method reduces amplification bias and chimera formation.Our data show that this method is suitable for as low as 10 pg input DNA, and offers the possibility of sequencing the parasite genome from small blood samples.
Affiliation: Wellcome Trust Sanger Institute, Hinxton, UK email@example.com.Show MeSH
Related in: MedlinePlus
Mentions: Non-coding regions of P. falciparum DNA contain ∼90% A+T base composition. Amplification of AT-rich genomes is a challenge to almost all commercially available polymerases. We have previously shown that addition of TMAC improves coverage of low GC regions of the genome during PCR,22 but the same has not been tested with φ29, the MDA polymerase. To investigate the effect of TMAC on MDA, we amplified P. falciparum 3D7 genomic DNA of varied input amounts (0.1–2 ng) both in the presence and absence of TMAC. We compared the quantity and quality of both products and observed that, like many commercial PCR polymerases, φ29 is inhibited by TMAC at certain levels of concentration. We have determined 60 mM as a concentration that is non-inhibitory to the polymerase and optimal for WGA of an AT-biased genome. Although the quantity of the amplification product was higher in the absence (standard procedure) than in the presence of 60 mM TMAC (data not shown), the quality of MDA product, in terms of coverage and base composition, was improved in the optimized procedure where TMAC was added (Figs 2 and 3). As shown in Fig. 2, amplification using a standard protocol (Std) resulted in excessive bias in regions of low complexity. Inspection of the over-amplified regions reveals sequences of low complexity and numerous repeat patterns. We used a tandem repeat finder programme26 and revealed numerous sequences in tandem repeat conformation that may have affected amplification bias. The top three tandem repeats are provided in Supplementary Table S1. Unlike the standard WGA protocol, our optimized amplification procedure abolished excessive amplification bias and produced a more uniform coverage. The mechanism by which this bias is corrected is not clear, but it is conceivable that TMAC stabilizes and stiffens the DNA backbone, thereby minimizing cis-priming by the looping of displaced DNA strands during amplification. We also show that base composition bias increased with a decrease in the amount of input DNA during amplification with standard protocols. This bias was not observed in samples amplified with the optimized (Opt) conditions (Fig. 3).Figure 2.
Affiliation: Wellcome Trust Sanger Institute, Hinxton, UK firstname.lastname@example.org.