Genomic DNA samples (10 ng) were denatured using heat (95°C) or the standard REPLI-g Kit alkaline lysis protocol. After amplification using Phi 29 polymerase, the CT values of 2 loci were compared between samples. The low CT values of loci amplified using the REPLI-g Kit alkaline lysis protocol indicate better locus representation, meaning there has been no loss of sequence information at these loci.
Real-time PCR of REPLI-g amplified DNA samples stored in 4 different formats at –20°C for the indicated time periods. Two loci, [A] locus A and [B] locus B, were assayed for each sample. gDNA: genomic DNA not amplified with REPLI-g. Storage formats: 50 µl REPLI-g reactions: 1) without further manipulation ("50 µl REPLI-g"); 2) aliquoted to 5 µl volumes ("5 µl REPLI-g"); 3) purified with QIAamp Mini Kit ("50 µl QIAamp purified REPLI-g"); and 4) diluted to a concentration of 50 ng/µl (50 µl diluted REPLI-g").
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. In subsequent real-time PCR, no bacterial DNA was detectable following the UV decontamination procedure.
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. [A] Comparable sequence coverage was observed for gDNA and REPLI-g Single Cell amplified DNA*. [B] 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).
Comprehensive analysis of 267 loci across the entire genome 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.
The REPLI-g Single Cell Kit or kits from Suppliers G, N, and S were used to individually amplify 5 human cells. [A] 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 WGA delivered unbiased amplification of DNA in each of the 5 cells, indicated by equivalent CT values for each marker. [B] 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.
Two easy-to-follow procedures enable WGA from 1–1000 cells or genomic DNA. Whole genome amplification using the REPLI-g Single Cell Kit involves 3 simple steps, regardless of whether 1–1000 cells (Protocol 1) or 10 ng genomic DNA (Protocol 2) is used as a starting material. First, the sample undergoes gentle alkaline denaturation, avoiding fragmentation and damage of template DNA. Next the sample is neutralized, and finally, incubated with the REPLI-g Single Cell master mix at 30°C. Regardless of your starting material, the REPLI-g Single Cell Kit delivers high yields of high-quality whole genome amplified DNA.
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.
[A] Upon encountering secondary DNA structures, Taq polymerase may pause synthesis, slip, or dissociate from the template. This can result in inaccurate DNA amplification, incomplete loci coverage, and short fragment sizes. [B] REPLI-g Kits utilize Phi29 polymerase, which displaces secondary structures enabling accurate and highly uniform amplification of the entire genome.
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.