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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

Determining the lowest threshold of input DNA mass for WGA. Different amounts of P. falciparum 3D7 genomic DNA (ranging from 2 ng to 100 fg, as shown in sample names) were used as an input template for amplification by Repli_g following the standard or optimized procedure. Samples were multiplexed and sequenced using a fast turn-around Illumina Miseq machine. Sequence reads mapped to the reference were normalized and analysed for coverage and base quality using the ‘CallableLoci’ program of GATK. A non-WGA sample was used as an unamplified control. Input quantities ranging from 2 ng down to 10 pg produced reads with high-quality ‘callable’ bases covering over 60% of the genome. Input DNA below the10-pg threshold produced poor base quality with only <30% of genome covered with ‘callable’ bases. Input DNA <10 pg showed a sharp increase in positions with zero coverage (bases uncovered) and an increase in gap sizes in genome coverage.
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DSU028F5: Determining the lowest threshold of input DNA mass for WGA. Different amounts of P. falciparum 3D7 genomic DNA (ranging from 2 ng to 100 fg, as shown in sample names) were used as an input template for amplification by Repli_g following the standard or optimized procedure. Samples were multiplexed and sequenced using a fast turn-around Illumina Miseq machine. Sequence reads mapped to the reference were normalized and analysed for coverage and base quality using the ‘CallableLoci’ program of GATK. A non-WGA sample was used as an unamplified control. Input quantities ranging from 2 ng down to 10 pg produced reads with high-quality ‘callable’ bases covering over 60% of the genome. Input DNA below the10-pg threshold produced poor base quality with only <30% of genome covered with ‘callable’ bases. Input DNA <10 pg showed a sharp increase in positions with zero coverage (bases uncovered) and an increase in gap sizes in genome coverage.

Mentions: Most studies with φ29 MDA have used an input DNA of ≥10 ng for WGA.19,20 In many cases, this amount may be difficult to obtain from valuable clinical specimens. We set to find out the minimal amount of parasite DNA that can be successfully amplified by the optimized conditions to obtain uniform genome coverage. We performed WGA on P. falciparum genomic DNA with an input amount ranging from 2 ng down to 100 femtograms (fg). WGA products were multiplexed and sequenced using Illumina MiSeq or HiSeq 2500 machines. Sequence reads generated were analysed to determine the minimum threshold required to produce optimal coverage suitable for various whole-genome studies including genotyping by SNP analysis. To assess the quality of the sequence data generated from each input amount, reads were mapped to the reference genome using BWA. CallableLoci program of the genome analyser tool kit (GATK)28 was used to inspect and count the proportion of the genome with high-quality base coverage (callable bases), positions of the genome with zero coverage (uncovered bases) and the size of coverage gaps. Using these metrics, we show that the number of callable loci (high-quality bases) remained relatively high for samples with input DNA ranging from 2 ng to 10 pg. However, the quality of sequence data dropped sharply for samples with input DNA <10 pg (Fig. 5). A similar trend was observed with the proportion of gap sizes (length of uncovered bases, Fig. 5 bottom panel) and chimeric reads (Fig. 4), where a sharp increase in these values was observed for samples with input DNA <10 pg. From these observations, we concluded that 10 pg is the minimal amount of P. falciparum input DNA that produces quality genome coverage under the optimized MDA conditions described. This amount of DNA is equivalent to only ∼380 parasite genomes.Figure 5.


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)

Determining the lowest threshold of input DNA mass for WGA. Different amounts of P. falciparum 3D7 genomic DNA (ranging from 2 ng to 100 fg, as shown in sample names) were used as an input template for amplification by Repli_g following the standard or optimized procedure. Samples were multiplexed and sequenced using a fast turn-around Illumina Miseq machine. Sequence reads mapped to the reference were normalized and analysed for coverage and base quality using the ‘CallableLoci’ program of GATK. A non-WGA sample was used as an unamplified control. Input quantities ranging from 2 ng down to 10 pg produced reads with high-quality ‘callable’ bases covering over 60% of the genome. Input DNA below the10-pg threshold produced poor base quality with only <30% of genome covered with ‘callable’ bases. Input DNA <10 pg showed a sharp increase in positions with zero coverage (bases uncovered) and an increase in gap sizes in genome coverage.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263299&req=5

DSU028F5: Determining the lowest threshold of input DNA mass for WGA. Different amounts of P. falciparum 3D7 genomic DNA (ranging from 2 ng to 100 fg, as shown in sample names) were used as an input template for amplification by Repli_g following the standard or optimized procedure. Samples were multiplexed and sequenced using a fast turn-around Illumina Miseq machine. Sequence reads mapped to the reference were normalized and analysed for coverage and base quality using the ‘CallableLoci’ program of GATK. A non-WGA sample was used as an unamplified control. Input quantities ranging from 2 ng down to 10 pg produced reads with high-quality ‘callable’ bases covering over 60% of the genome. Input DNA below the10-pg threshold produced poor base quality with only <30% of genome covered with ‘callable’ bases. Input DNA <10 pg showed a sharp increase in positions with zero coverage (bases uncovered) and an increase in gap sizes in genome coverage.
Mentions: Most studies with φ29 MDA have used an input DNA of ≥10 ng for WGA.19,20 In many cases, this amount may be difficult to obtain from valuable clinical specimens. We set to find out the minimal amount of parasite DNA that can be successfully amplified by the optimized conditions to obtain uniform genome coverage. We performed WGA on P. falciparum genomic DNA with an input amount ranging from 2 ng down to 100 femtograms (fg). WGA products were multiplexed and sequenced using Illumina MiSeq or HiSeq 2500 machines. Sequence reads generated were analysed to determine the minimum threshold required to produce optimal coverage suitable for various whole-genome studies including genotyping by SNP analysis. To assess the quality of the sequence data generated from each input amount, reads were mapped to the reference genome using BWA. CallableLoci program of the genome analyser tool kit (GATK)28 was used to inspect and count the proportion of the genome with high-quality base coverage (callable bases), positions of the genome with zero coverage (uncovered bases) and the size of coverage gaps. Using these metrics, we show that the number of callable loci (high-quality bases) remained relatively high for samples with input DNA ranging from 2 ng to 10 pg. However, the quality of sequence data dropped sharply for samples with input DNA <10 pg (Fig. 5). A similar trend was observed with the proportion of gap sizes (length of uncovered bases, Fig. 5 bottom panel) and chimeric reads (Fig. 4), where a sharp increase in these values was observed for samples with input DNA <10 pg. From these observations, we concluded that 10 pg is the minimal amount of P. falciparum input DNA that produces quality genome coverage under the optimized MDA conditions described. This amount of DNA is equivalent to only ∼380 parasite genomes.Figure 5.

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