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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

Boxplot of in silico genotyping concordance analysing. A total of 20,737 high-quality SNP positions were genotyped in both WGA and non-WGA samples. Call comparison was performed between matched pairs of corresponding WGA and non-WGA datasets and results were grouped into ‘Perfect Concordance’, ‘WGA Missing Alleles’, ‘WGA New Alleles’ and ‘Undetermined’. Perfect_Concordance (identical alleles) had a median of 97.95% (range 83.11–99.58%; n = 17). A median of 0.48% calls represent alleles that were called in the WGA samples, but not in the non-WGA samples (WGA_New_Alleles). A median of 0.94% calls were present in non-WGA, but absent in WGA, samples (WGA_Missing_Alleles). The proportion of calls that were missing (Undetermined) had a median of 0.96% (Supplementary Table S4).
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DSU028F6: Boxplot of in silico genotyping concordance analysing. A total of 20,737 high-quality SNP positions were genotyped in both WGA and non-WGA samples. Call comparison was performed between matched pairs of corresponding WGA and non-WGA datasets and results were grouped into ‘Perfect Concordance’, ‘WGA Missing Alleles’, ‘WGA New Alleles’ and ‘Undetermined’. Perfect_Concordance (identical alleles) had a median of 97.95% (range 83.11–99.58%; n = 17). A median of 0.48% calls represent alleles that were called in the WGA samples, but not in the non-WGA samples (WGA_New_Alleles). A median of 0.94% calls were present in non-WGA, but absent in WGA, samples (WGA_Missing_Alleles). The proportion of calls that were missing (Undetermined) had a median of 0.96% (Supplementary Table S4).

Mentions: Using a list of high-quality SNP positions and alleles from the genetic crosses, we performed in silico genotyping of both the WGA and non-WGA samples on 20,737 positions, counting alleles present in at least five reads. Call comparisons between the WGA and non-WGA from same samples were grouped into ‘Perfect Concordance (identical)’, ‘WGA Missing Alleles’, ‘WGA New Alleles’ and ‘Undetermined’ (missing allele). Identical alleles with perfect concordance between WGA and non-WGA samples had a median of 97.95% (Fig. 6). A median of 0.48% calls represent alleles that were called in the WGA, but not in the non-WGA, samples. These reflect cases where the WGA sample had a mixed call and the non-WGA sample had a single-allele call. A median of 0.94% represent alleles that were missing in WGA, but were called in non-WGA samples. Discordant single-allele calls were extremely rare, with a median of zero and a mean of 2.9 SNPs per sample. Four of the 17 samples showed such discordant calls, and only three samples had more than one such call. The 3D7 (3D7_Glasgow) reference sample showed 99.58% identical calls, and only one new allele in WGA sample was returned as a mixed call (Supplementary Table S4).Figure 6.


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)

Boxplot of in silico genotyping concordance analysing. A total of 20,737 high-quality SNP positions were genotyped in both WGA and non-WGA samples. Call comparison was performed between matched pairs of corresponding WGA and non-WGA datasets and results were grouped into ‘Perfect Concordance’, ‘WGA Missing Alleles’, ‘WGA New Alleles’ and ‘Undetermined’. Perfect_Concordance (identical alleles) had a median of 97.95% (range 83.11–99.58%; n = 17). A median of 0.48% calls represent alleles that were called in the WGA samples, but not in the non-WGA samples (WGA_New_Alleles). A median of 0.94% calls were present in non-WGA, but absent in WGA, samples (WGA_Missing_Alleles). The proportion of calls that were missing (Undetermined) had a median of 0.96% (Supplementary Table S4).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DSU028F6: Boxplot of in silico genotyping concordance analysing. A total of 20,737 high-quality SNP positions were genotyped in both WGA and non-WGA samples. Call comparison was performed between matched pairs of corresponding WGA and non-WGA datasets and results were grouped into ‘Perfect Concordance’, ‘WGA Missing Alleles’, ‘WGA New Alleles’ and ‘Undetermined’. Perfect_Concordance (identical alleles) had a median of 97.95% (range 83.11–99.58%; n = 17). A median of 0.48% calls represent alleles that were called in the WGA samples, but not in the non-WGA samples (WGA_New_Alleles). A median of 0.94% calls were present in non-WGA, but absent in WGA, samples (WGA_Missing_Alleles). The proportion of calls that were missing (Undetermined) had a median of 0.96% (Supplementary Table S4).
Mentions: Using a list of high-quality SNP positions and alleles from the genetic crosses, we performed in silico genotyping of both the WGA and non-WGA samples on 20,737 positions, counting alleles present in at least five reads. Call comparisons between the WGA and non-WGA from same samples were grouped into ‘Perfect Concordance (identical)’, ‘WGA Missing Alleles’, ‘WGA New Alleles’ and ‘Undetermined’ (missing allele). Identical alleles with perfect concordance between WGA and non-WGA samples had a median of 97.95% (Fig. 6). A median of 0.48% calls represent alleles that were called in the WGA, but not in the non-WGA, samples. These reflect cases where the WGA sample had a mixed call and the non-WGA sample had a single-allele call. A median of 0.94% represent alleles that were missing in WGA, but were called in non-WGA samples. Discordant single-allele calls were extremely rare, with a median of zero and a mean of 2.9 SNPs per sample. Four of the 17 samples showed such discordant calls, and only three samples had more than one such call. The 3D7 (3D7_Glasgow) reference sample showed 99.58% identical calls, and only one new allele in WGA sample was returned as a mixed call (Supplementary Table S4).Figure 6.

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