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Improved multiple displacement amplification (iMDA) and ultraclean reagents.

Motley ST, Picuri JM, Crowder CD, Minich JJ, Hofstadler SA, Eshoo MW - BMC Genomics (2014)

Bottom Line: To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed.The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA.The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

View Article: PubMed Central - PubMed

Affiliation: Ibis Biosciences an Abbott Company, 2251 Faraday Ave, Suite 150, Carlsbad, CA 92008, USA. Mark.eshoo@abbott.com.

ABSTRACT

Background: Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).

Results: A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.

Conclusions: The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

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Lorenz curve iMDA and genomic sequencing ofB.cereus. Lorentz curves depict the relative bias in average read coverage across the B. cereus genome. Each curve was calculated by dividing the genome into 500 bp bins, counting the average read depth across each bin, and using the resultant cumulative distribution function for read depth to determine the cumulative proportion of total genome coverage (y-axis) accounted for by the cumulative proportion of bins (x-axis). The ideal Lorentz curve (black line) for a distribution in which all of the bins have the same coverage is plotted for comparison.
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Fig4: Lorenz curve iMDA and genomic sequencing ofB.cereus. Lorentz curves depict the relative bias in average read coverage across the B. cereus genome. Each curve was calculated by dividing the genome into 500 bp bins, counting the average read depth across each bin, and using the resultant cumulative distribution function for read depth to determine the cumulative proportion of total genome coverage (y-axis) accounted for by the cumulative proportion of bins (x-axis). The ideal Lorentz curve (black line) for a distribution in which all of the bins have the same coverage is plotted for comparison.

Mentions: In order to assess coverage uniformity and relative bias generated by the iMDA process we generated Lorenz curves from sequence derived from 1 pg of B.cereus genomic template amplified by iMDA and unamplified template. The results are shown in Figure 4 in which we compare the Lorenz curves from both samples at an average coverage depth of 73X. The diagonal line indicates perfect uniformity of coverage and deviation indicates an uneven distribution of reads. It is evident that the iMDA provides a very high uniformity of genomic coverage and this is in good agreement with the 99.9% coverage value calculated in Table 2.Figure 4


Improved multiple displacement amplification (iMDA) and ultraclean reagents.

Motley ST, Picuri JM, Crowder CD, Minich JJ, Hofstadler SA, Eshoo MW - BMC Genomics (2014)

Lorenz curve iMDA and genomic sequencing ofB.cereus. Lorentz curves depict the relative bias in average read coverage across the B. cereus genome. Each curve was calculated by dividing the genome into 500 bp bins, counting the average read depth across each bin, and using the resultant cumulative distribution function for read depth to determine the cumulative proportion of total genome coverage (y-axis) accounted for by the cumulative proportion of bins (x-axis). The ideal Lorentz curve (black line) for a distribution in which all of the bins have the same coverage is plotted for comparison.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4061449&req=5

Fig4: Lorenz curve iMDA and genomic sequencing ofB.cereus. Lorentz curves depict the relative bias in average read coverage across the B. cereus genome. Each curve was calculated by dividing the genome into 500 bp bins, counting the average read depth across each bin, and using the resultant cumulative distribution function for read depth to determine the cumulative proportion of total genome coverage (y-axis) accounted for by the cumulative proportion of bins (x-axis). The ideal Lorentz curve (black line) for a distribution in which all of the bins have the same coverage is plotted for comparison.
Mentions: In order to assess coverage uniformity and relative bias generated by the iMDA process we generated Lorenz curves from sequence derived from 1 pg of B.cereus genomic template amplified by iMDA and unamplified template. The results are shown in Figure 4 in which we compare the Lorenz curves from both samples at an average coverage depth of 73X. The diagonal line indicates perfect uniformity of coverage and deviation indicates an uneven distribution of reads. It is evident that the iMDA provides a very high uniformity of genomic coverage and this is in good agreement with the 99.9% coverage value calculated in Table 2.Figure 4

Bottom Line: To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed.The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA.The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

View Article: PubMed Central - PubMed

Affiliation: Ibis Biosciences an Abbott Company, 2251 Faraday Ave, Suite 150, Carlsbad, CA 92008, USA. Mark.eshoo@abbott.com.

ABSTRACT

Background: Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).

Results: A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.

Conclusions: The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

Show MeSH