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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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Metagenomic sequence analysis of 10 fg ofB. cereusgenomic DNA amplified by (a) the Ibis Ultraclean iMDA protocol or with commercial WGA Kits (b) Genomiphi V2 WGA or (c) Qiagen REPLI-g WGA. All amplified reactions were sequenced by ion semiconductor sequencing (Ion Torrent PGM) followed by metagenomic analysis. *All reads that mapped to the B. cereus clade (i.e., B. cereus, B. thuringiensis, and B. anthracis) are reported as B. cereus. **All reads that mapped to Escherichia or Shigella are reported as E. coli.
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Fig1: Metagenomic sequence analysis of 10 fg ofB. cereusgenomic DNA amplified by (a) the Ibis Ultraclean iMDA protocol or with commercial WGA Kits (b) Genomiphi V2 WGA or (c) Qiagen REPLI-g WGA. All amplified reactions were sequenced by ion semiconductor sequencing (Ion Torrent PGM) followed by metagenomic analysis. *All reads that mapped to the B. cereus clade (i.e., B. cereus, B. thuringiensis, and B. anthracis) are reported as B. cereus. **All reads that mapped to Escherichia or Shigella are reported as E. coli.

Mentions: The sensitivity and specificity of the iMDA protocol was compared to sensitivity and specificity of two different commercial MDA kits: Qiagen REPLI-g® UltraFast Mini Kit and GenomiPhi V2 DNA Amplification Kit. For these tests, a very low level of B. cereus bacterial genomic DNA (10 fg, equivalent to the expected DNA from ~1-2 bacterial cells) was used as a template in the amplification reactions. The resulting amplified DNA was sequenced on an Ion Torrent semiconductor sequencing system. The DNA sequences from each reaction were subject to analysis with the Ibis Galaxy Analysis software to determine their meta-genomic species composition; the identified species in each sample were used to construct the pie-charts shown in Figure 1. The iMDA sequencing reactions produced 1.14 × 106 reads (average read length of 118 bases) with >1.13 × 106 of the reads specifically identified by the Ibis Galaxy analysis. The iMDA template genome, B. cereus was identified as the source of >99.4% (B. cereus specific reads/total mapped reads) of the mapped reads with 0.2% of the reads mapping to other Bacillus clade species.Figure 1


Improved multiple displacement amplification (iMDA) and ultraclean reagents.

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

Metagenomic sequence analysis of 10 fg ofB. cereusgenomic DNA amplified by (a) the Ibis Ultraclean iMDA protocol or with commercial WGA Kits (b) Genomiphi V2 WGA or (c) Qiagen REPLI-g WGA. All amplified reactions were sequenced by ion semiconductor sequencing (Ion Torrent PGM) followed by metagenomic analysis. *All reads that mapped to the B. cereus clade (i.e., B. cereus, B. thuringiensis, and B. anthracis) are reported as B. cereus. **All reads that mapped to Escherichia or Shigella are reported as E. coli.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Metagenomic sequence analysis of 10 fg ofB. cereusgenomic DNA amplified by (a) the Ibis Ultraclean iMDA protocol or with commercial WGA Kits (b) Genomiphi V2 WGA or (c) Qiagen REPLI-g WGA. All amplified reactions were sequenced by ion semiconductor sequencing (Ion Torrent PGM) followed by metagenomic analysis. *All reads that mapped to the B. cereus clade (i.e., B. cereus, B. thuringiensis, and B. anthracis) are reported as B. cereus. **All reads that mapped to Escherichia or Shigella are reported as E. coli.
Mentions: The sensitivity and specificity of the iMDA protocol was compared to sensitivity and specificity of two different commercial MDA kits: Qiagen REPLI-g® UltraFast Mini Kit and GenomiPhi V2 DNA Amplification Kit. For these tests, a very low level of B. cereus bacterial genomic DNA (10 fg, equivalent to the expected DNA from ~1-2 bacterial cells) was used as a template in the amplification reactions. The resulting amplified DNA was sequenced on an Ion Torrent semiconductor sequencing system. The DNA sequences from each reaction were subject to analysis with the Ibis Galaxy Analysis software to determine their meta-genomic species composition; the identified species in each sample were used to construct the pie-charts shown in Figure 1. The iMDA sequencing reactions produced 1.14 × 106 reads (average read length of 118 bases) with >1.13 × 106 of the reads specifically identified by the Ibis Galaxy analysis. The iMDA template genome, B. cereus was identified as the source of >99.4% (B. cereus specific reads/total mapped reads) of the mapped reads with 0.2% of the reads mapping to other Bacillus clade species.Figure 1

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