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Optimized whole-genome amplification strategy for extremely AT-biased template.

Oyola SO, Manske M, Campino S, Claessens A, Hamilton WL, Kekre M, Drury E, Mead D, Gu Y, Miles A, MacInnis B, Newbold C, Berriman M, Kwiatkowski DP - DNA Res. (2014)

Bottom Line: We present a strategy for whole-genome amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing.We have developed WGA conditions that incorporate tetramethylammonium chloride for improved amplification and coverage of AT-rich regions of the genome.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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Hinxton, UK so1@sanger.ac.uk.

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Related in: MedlinePlus

Bar graph of chimeric read analysis. Different amounts of P. falciparum 3D7 genomic DNA (ranging from 2 ng to 100 fg as shown in sample labels) were used as an input template for amplification by Repli_g following the standard or optimized procedure. Amplification products were sequenced as PCR-free, and reads obtained were normalized and analysed for the presence of chimeric reads. A non-WGA sample (control) showed the least number of chimers. Using the standard procedure, the number of chimers increased with a decrease in the amount of input DNA. Samples amplified using the optimized procedure showed a decrease in chimer formation that remained low and steady from 2 ng to 100 pg of input DNA.
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DSU028F4: Bar graph of chimeric read analysis. Different amounts of P. falciparum 3D7 genomic DNA (ranging from 2 ng to 100 fg as shown in sample labels) were used as an input template for amplification by Repli_g following the standard or optimized procedure. Amplification products were sequenced as PCR-free, and reads obtained were normalized and analysed for the presence of chimeric reads. A non-WGA sample (control) showed the least number of chimers. Using the standard procedure, the number of chimers increased with a decrease in the amount of input DNA. Samples amplified using the optimized procedure showed a decrease in chimer formation that remained low and steady from 2 ng to 100 pg of input DNA.

Mentions: Formation of chimeric reads is a major problem with MDA technology.27 Chimeras cause serious mapping and assembly problems, and therefore reduce the quality and quantity of the WGA product. We analysed the formation of chimeras by comparing WGA products following standard and optimized procedures. The number of chimeric reads increased as the amount of input DNA was reduced (Fig. 4). However, optimizing WGA condition by including TMAC additive decreased the formation of chimeras and improved the quality of reads in P. falciparum WGA.Figure 4.


Optimized whole-genome amplification strategy for extremely AT-biased template.

Oyola SO, Manske M, Campino S, Claessens A, Hamilton WL, Kekre M, Drury E, Mead D, Gu Y, Miles A, MacInnis B, Newbold C, Berriman M, Kwiatkowski DP - DNA Res. (2014)

Bar graph of chimeric read analysis. Different amounts of P. falciparum 3D7 genomic DNA (ranging from 2 ng to 100 fg as shown in sample labels) were used as an input template for amplification by Repli_g following the standard or optimized procedure. Amplification products were sequenced as PCR-free, and reads obtained were normalized and analysed for the presence of chimeric reads. A non-WGA sample (control) showed the least number of chimers. Using the standard procedure, the number of chimers increased with a decrease in the amount of input DNA. Samples amplified using the optimized procedure showed a decrease in chimer formation that remained low and steady from 2 ng to 100 pg of input DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263299&req=5

DSU028F4: Bar graph of chimeric read analysis. Different amounts of P. falciparum 3D7 genomic DNA (ranging from 2 ng to 100 fg as shown in sample labels) were used as an input template for amplification by Repli_g following the standard or optimized procedure. Amplification products were sequenced as PCR-free, and reads obtained were normalized and analysed for the presence of chimeric reads. A non-WGA sample (control) showed the least number of chimers. Using the standard procedure, the number of chimers increased with a decrease in the amount of input DNA. Samples amplified using the optimized procedure showed a decrease in chimer formation that remained low and steady from 2 ng to 100 pg of input DNA.
Mentions: Formation of chimeric reads is a major problem with MDA technology.27 Chimeras cause serious mapping and assembly problems, and therefore reduce the quality and quantity of the WGA product. We analysed the formation of chimeras by comparing WGA products following standard and optimized procedures. The number of chimeric reads increased as the amount of input DNA was reduced (Fig. 4). However, optimizing WGA condition by including TMAC additive decreased the formation of chimeras and improved the quality of reads in P. falciparum WGA.Figure 4.

Bottom Line: We present a strategy for whole-genome amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing.We have developed WGA conditions that incorporate tetramethylammonium chloride for improved amplification and coverage of AT-rich regions of the genome.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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Hinxton, UK so1@sanger.ac.uk.

Show MeSH
Related in: MedlinePlus