NGS using Whole Genome Amplified DNA

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Reliable NGS results from limited starting materials
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DNA sequence analysis and genotyping of biological samples using innovative instrumentation, such as next-generation sequencing (NGS) platforms, is often limited by the small amount of sample available. With REPLI-g technology, WGA on just single cells or non-degraded purified DNA delivers NGS results that are comparable to those obtained with unamplified gDNA.
REPLI-g amplified DNA has been successfully used in NGS
Next-generation sequencing single cells
NGS citations that use REPLI-g
REPLI-g amplified DNA has been successfully used in next-generation sequencing
Numerous publications have demonstrated the successful utilization of REPLI-g amplified DNA for next-generation sequencing (NGS) applications that range from exome and whole genome sequencing of tumor cells, to metagenomics research, to single cell analysis (see table).

Since the use of whole genome amplified DNA for NGS and array applications has been debated, we detected potential factors that could influence the success of using amplified DNA for these downstream applications. We determined that the quality of input material strongly influences the success of downstream NGS experiments. If working with low-quality DNA (e.g., degraded DNA) or aged tissue material, the resulting amplified DNA may not give reliable results (data not shown). However, WGA, using REPLI-g technology, on intact cells or non-degraded purified DNA, shows that NGS results are comparable to those obtained with purified gDNA. Sequence coverage and alignment comparison of the genomic loci sequence indicates minimized levels of junk DNA after WGA, whereas error rates are in a similar percentage range for both amplified and genomic DNA (see figure Comparable NGS results obtained using purified gDNA or REPLI-g Midi amplified DNA).

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Next-generation sequencing from just single cells
The REPLI-g Single Cell Kit is specially designed to uniformly amplify genomic DNA (gDNA) directly from single cells (<1000 cells to as little as 1 bacterial or tumor cell) or purified gDNA, with negligible sequence bias and maximized genome coverage, making the REPLI-g Single Cell Kit an excellent tool for a wide range of NGS applications, including cancer research, metagenomics, and stem cell biology.

DNA amplified using the REPLI-g Single Cell Kit has been tested with, and is highly suited for, numerous downstream analyses, including next-generation sequencing. Since there is no requirement for a separate PCR-based amplification step, REPLI-g whole genome amplification and library preparation requires less hands-on time and results in longer read-lengths than PCR-based methods (see figure Less hands-on time and more sequence information). High-quality, comparable NGS results showing a high percentage of sequence coverage and very low error rates are achieved with both purified genomic DNA or REPLI-g Single Cell amplified DNA, including when starting from just a single bacterial cell (see figure Comparable NGS results). These findings are underscored by a comprehensive analysis of a wide range of markers covering all human autosomal chromosomes and the X chromosome, with 3 different independent experiments demonstrating that DNA is successfully amplified from all areas of the genome without a single drop-out (see figure Complete genome coverage and Unbiased DNA amplification from a single cell).

Unbiased amplification from a single cell is achieved with Multiple Displacement Amplification (MDA) technology and a modified form of Phi29 Polymerase (see figure Multiple Displacement Amplification (MDA) technology), along with a unique, controlled decontamination procedure to avoid amplification of contaminating DNA, ensuring highly reliable results every time (see figure Innovative UV treatment). 

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NGS citations that use REPLI-g
Numerous publications have demonstrated the successful utilization of REPLI-g amplified DNA for NGS applications, ranging from exome and whole genome sequencing of tumor cells, to metagenomics research, to single cell analysis.

Example citations for the successful use of REPLI-g amplified DNA in downstream NGS analysis
REPLI-g Kit Year Research topic Organism Downstream analysis Title Citation
REPLI-g Mini Kit  2012   Somatic mutations Eukaryotes/Homo sapiens Illumina HiSeq Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Hou, Y. et al. (2012) Cell 148, 873.
 2011 Congenital disease Eukaryotes/Homo sapiens Resequencing array (Affymetrix) High quality DNA sequence capture of 524 disease candidate genes. Shen, P. et al. (2011) Proc. Natl. Acad. Sci. USA 108, 6549.
 2010 Metagenomics Eukaryotes/Coryno-carpus Illumina GAII Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform. Atherton, R.A. et al. (2010) Plant Methods 6, 22.
 2010 Methodology report Eukaryotes/Homo sapiens SOLiD 3 (Life Technologies) Semi-automated library preparation for high-throughput DNA sequencing platforms. Farias-Hesson, E., et al. (2010) J. Biomed Biotechnol 2010, 617469.
 2008 Metagenomics Prokaryotes Roche 454 Metagenomic signatures of the Peru Margin subseafloor biosphere show a genetically distinct environment. Biddle, J.F. et al. (2008) Proc. Natl. Acad. Sci. USA 105,10583
REPLI-g Midi Kit  2012 Metagenomics Prokaryotes/Clostri-diaceae Roche 454 Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB). Pamp, S.J. et al. (2012) Genome Res. 22, 1107.
 2012 Synthesis of non-methylated DNA Prokaryotes PacBio RS (Pacific Biosciences) Characterization of DNA methyltrasferase specificities using single-molecule, real-time DNA sequencing. Clark, T.A., et al. (2012) Nucleic Acids Research 40, e29.
 2012 Tropical diseases Eukaryotes/Schisto-soma Illumina GAII Whole-genome sequence of Schistosoma haematobium. Young, N.D., et al. (2012) Nat. Genet. 44, 221.
 2011 Parasitic diseases Eukaryotes/Ascaris Illumina HiSeq Ascaris suum draft genome. Jex, A.R., et al. (2011) Nature 479, 529.
REPLI-g Screening Kit  2011 Genome analysis Prokaryotes/Clostri-diaceae Roche 454 Filtering “genic” open reading frames from genomic DNA samples for advanced annotation. D’Angelo, S., et al. (2011) BMC Genomics 12 Suppl. 1, S5.
Not specified  2011 Metagenomics Eukaryotes/Plasmo-dium Illumina GAII/Illumina HiSeq Hybrid selection for sequencing pathogen genomes from clinical samples. Melnikov, A., et al (2011) Genome Biol. 12, R73.
 2011 Congenital diseases Eukaryotes/Homo sapiens Roche 454/Illumina GAII Identification of disease-causing mutations in autosomal dominant retinitis pigmentosa (adRP) using next-generation DNA sequencing. Bowne, S.J., et al. (2011) Invest. Ophthalmol. Vis. Sci. 52, 494.


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REPLI-g Single Cell Kit (24)
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For whole genome amplification from single cells, limited samples, or purified genomic DNA
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Comparable NGS results using genomic or single-cell amplified DNA
Comparable NGS results obtained using purified genomic DNA or REPLI-g Single Cell amplified DNA.
Whole genome sequencing of the Bacillus subtilis genome was performed on the Illumina MiSeq instrument. For analysis, 2 μg of genomic DNA or DNA amplified from a single cell (three different single cell experiments) and 103 cells, using the REPLI-g Single Cell Kit, was sheared into 300 bp fragments and 1 μg of each was used for library preparation. Comparable sequence coverage was observed for gDNA and REPLI-g Single Cell amplified DNA*. Comparison of nonamplified and REPLI-g amplified DNA revealed error rates in a similar, very low, percentage range.
* Aligned using the Burrows-Wheeler Alignment program (cut-off: 10x coverage): bio-bwa.sourceforge.net.
Comparison on non-amplified and REPLI-g Single Cell amplified DNA also revealed that sequences mapped to the genome with high percentage rates (data not shown
Comparable NGS results obtained using gDNA or REPLI-g amplified DNA
Comparable NGS results obtained using gDNA or REPLI-g Midi amplified DNA.
Whole genome sequencing of the Bacillus subtilis genome was performed. For analysis, 2 μg of genomic DNA or DNA amplified from 105 cells using the REPLI-g Midi Kit was sheared into 300 bp fragments. For library preparation, 1 μg of each was used. Sequencing was performed on the Illumina MiSeq instrument. (A) Comparable sequence coverage was observed for both gDNA and REPLI-g amplified DNA. (B) Alignment comparison of the genomic loci sequence demonstrates comparably high percentage of alignment for REPLI-g amplified DNA in comparison to the gDNA, which is an indication of minimized levels of junk DNA after WGA (whole genome amplification). Comparison of nonamplified and REPLI-g amplified DNA revealed error rates (mismatch, high-quality error, indels, or chimeras) in a similar percentage range. (Alignment comparison performed using SMALT [Welcome Trust Sanger Institute]).
Complete genome coverage.
Complete genome coverage.
Comprehensive analysis of 267 loci spread out over different chromosomes across the entire human genome (as indicated) was performed using RT2 qPCR Primer Assays (QIAGEN) and real-time PCR following DNA amplification with the REPLI-g Single Cell Kit from 3 different single cell experiments. Low and consistent CT values, with no dropout from any marker, indicate that DNA was successfully amplified from all areas of the genome and is highly suited for single-cell genomics.
Innovative UV treatment eliminates any detectable trace of residual DNA in kit components
Innovative UV treatment.
Bacterial DNA (2000 copies) was spiked into REPLI-g sc Reaction Buffer, which was then irradiated with UV using the standard procedure for all buffers and reagents provided with the REPLI-g Single Cell Kit (following UV treatment, the kits undergo stringent quality control to ensure complete functionality). In subsequent real-time PCR, no bacterial DNA was detectable following the UV decontamination procedure.
Next-generation sequencing using REPLI-g amplified DNA requires less hands-on time and generates more sequence information than PCR-based methods
Less hands-on time and more sequence information.
PCR-based whole genome amplification (WGA) and library preparation for next-generation sequencing requires a purification step prior to library preparation that can result in ~3 times more hands-on time than REPLI-g based WGA and library preparation. Additionally, unlike REPLI-g amplified DNA, PCR-based methods also include PCR primer binding sites (indicated in red) on the WGA amplification product. Since next-generation sequencing read-lengths are between 50–200 bp, the resulting genome coverage could be strongly reduced using a PCR-based WGA method.
Multiple Displacement Amplification (MDA) technology delivers long read lengths with isothermal amplification.
Multiple Displacement Amplification (MDA) technology delivers long read lengths with isothermal amplification.
Primers (arrows) anneal to the template DNA and are extended at 30°C by Phi 29 polymerase, which moves along the DNA template strand, displacing the complementary strand while becoming a template itself for replication. In contrast to PCR amplification, MDA does not require different temperatures and ends in very long fragments with low mutation rates.
The REPLI-g Single Cell Kit delivers unbiased amplification of DNA from a single cell
The REPLI-g Single Cell Kit delivers highly reliable, unbiased amplification of DNA from a single cell.
The REPLI-g Single Cell Kit or single cell kits from Suppliers G, N, and S were used to individually amplify 5 human cells. Real-time PCR was used to analyze 3 markers to identify loss or variability in the amount of genomic loci. Unlike kits from other suppliers, the REPLI-g Single Cell Kit delivered unbiased amplification of DNA in each of the 5 cells, indicated by equivalent CT values for each marker. Unlike with the REPLI-g Single Cell Kit, DNA amplified using the kits from Suppliers G and N demonstrated high dropout rates. For both kits, genomic marker X54 was not amplified in 2 of the 5 cells tested, and the kit from Supplier G did not amplify marker 99 in 1 of the 5 cells, indicating incomplete genome coverage and biased amplification that makes these kits unsuitable for reliable single cell research.