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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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Whole genome coverage of iMDA DNA. One picogram (pg) of B. cereus genomic DNA was amplified by the iMDA protocol and the amplified DNA was sequenced. Plots show depth of coverage vs. the position in the reference genome at increasing magnifications. (a) Mapping across the entire reference genome. (b) Mapping from 2 MB to 2.5 MB in the reference genome. (c) Mapping from 2.200 MB to 2.205 MB in the reference genome.
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Fig2: Whole genome coverage of iMDA DNA. One picogram (pg) of B. cereus genomic DNA was amplified by the iMDA protocol and the amplified DNA was sequenced. Plots show depth of coverage vs. the position in the reference genome at increasing magnifications. (a) Mapping across the entire reference genome. (b) Mapping from 2 MB to 2.5 MB in the reference genome. (c) Mapping from 2.200 MB to 2.205 MB in the reference genome.

Mentions: To assess whether the iMDA method achieves relatively complete coverage of the input genome, a non-stochastic amount of B. cereus template DNA (1 pg) was amplified (11,638,000 fold) in a 2-h iMDA reaction. An aliquot of the iMDA reaction and two independent non-amplified B. cereus genomic DNA (1 μg) samples were used to produce sequencing libraries. The sequencing reactions were mapped to the B. cereus ATCC 10987 (NC_003909.8) published sequence with NextGENe software from SoftGenetics. The results are summarized in Table 2. Sequence data obtained from the iMDA template and from both of the genomic templates covered more than 99.98% of the reference genome at least 1X coverage) and greater than 99.9% of the genome with at least 5X coverage. The average read lengths were comparable (212 to 224 bases) as were the total number of bases read (398 MB for the iMDA template vs. 574 MB for the genomic DNA). The average coverage of the iMDA template was 64 fold, whereas that of the purified genome was 105 fold. Figure 2 depicts the coverage vs. position for the iMDA data at various degrees of resolution; these data demonstrate the uniformity of the genomic coverage indicating that the iMDA protocol did not introduce bias. The depth of coverage between iMDA and genomic DNA samples were compared in 500-bp bins across the genome (Figure 3). The average variation between the two samples of genomic DNA was 1.1 fold (±1.1). The average variation between iMDA and the genomic DNA was 1.5 fold (±1.4); for 82.7% of the sequence bins the variation was within 2 fold and for 99.0% of the bins variation was within 4 fold. This is especially notable as the iMDA sample was amplified more than 1.1x107 fold. We were not able to make a comparable comparison for representation and coverage using commercial MDA kits. For example MDA utilizing the illustra genomiphi V2 kit with 1 pg of B cereus template DNA yielded only 37 ng of B. cereus DNA, as determined by qPCR. The total DNA yield of 10.9 μg of DNA indicated only 3.3x104 fold amplification of the B. cereus DNA and less than 1% of the total amplified DNA was derived from the template DNA.Table 2


Improved multiple displacement amplification (iMDA) and ultraclean reagents.

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

Whole genome coverage of iMDA DNA. One picogram (pg) of B. cereus genomic DNA was amplified by the iMDA protocol and the amplified DNA was sequenced. Plots show depth of coverage vs. the position in the reference genome at increasing magnifications. (a) Mapping across the entire reference genome. (b) Mapping from 2 MB to 2.5 MB in the reference genome. (c) Mapping from 2.200 MB to 2.205 MB in the reference genome.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Whole genome coverage of iMDA DNA. One picogram (pg) of B. cereus genomic DNA was amplified by the iMDA protocol and the amplified DNA was sequenced. Plots show depth of coverage vs. the position in the reference genome at increasing magnifications. (a) Mapping across the entire reference genome. (b) Mapping from 2 MB to 2.5 MB in the reference genome. (c) Mapping from 2.200 MB to 2.205 MB in the reference genome.
Mentions: To assess whether the iMDA method achieves relatively complete coverage of the input genome, a non-stochastic amount of B. cereus template DNA (1 pg) was amplified (11,638,000 fold) in a 2-h iMDA reaction. An aliquot of the iMDA reaction and two independent non-amplified B. cereus genomic DNA (1 μg) samples were used to produce sequencing libraries. The sequencing reactions were mapped to the B. cereus ATCC 10987 (NC_003909.8) published sequence with NextGENe software from SoftGenetics. The results are summarized in Table 2. Sequence data obtained from the iMDA template and from both of the genomic templates covered more than 99.98% of the reference genome at least 1X coverage) and greater than 99.9% of the genome with at least 5X coverage. The average read lengths were comparable (212 to 224 bases) as were the total number of bases read (398 MB for the iMDA template vs. 574 MB for the genomic DNA). The average coverage of the iMDA template was 64 fold, whereas that of the purified genome was 105 fold. Figure 2 depicts the coverage vs. position for the iMDA data at various degrees of resolution; these data demonstrate the uniformity of the genomic coverage indicating that the iMDA protocol did not introduce bias. The depth of coverage between iMDA and genomic DNA samples were compared in 500-bp bins across the genome (Figure 3). The average variation between the two samples of genomic DNA was 1.1 fold (±1.1). The average variation between iMDA and the genomic DNA was 1.5 fold (±1.4); for 82.7% of the sequence bins the variation was within 2 fold and for 99.0% of the bins variation was within 4 fold. This is especially notable as the iMDA sample was amplified more than 1.1x107 fold. We were not able to make a comparable comparison for representation and coverage using commercial MDA kits. For example MDA utilizing the illustra genomiphi V2 kit with 1 pg of B cereus template DNA yielded only 37 ng of B. cereus DNA, as determined by qPCR. The total DNA yield of 10.9 μg of DNA indicated only 3.3x104 fold amplification of the B. cereus DNA and less than 1% of the total amplified DNA was derived from the template DNA.Table 2

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
Related in: MedlinePlus