MDA in Capillary for Whole Genome Amplification
نویسندگان
چکیده
Whole genome amplification (WGA) plays an important role in sample preparation of low-input templates for high-throughput sequencing. Multiple displacement amplification (MDA), a popular isothermal WGA methods, suffers a major hurdle of highly uneven amplification. Optimizations have been made in the past by separating the reagents into numbers of tiny chambers or droplets in microfluidic devices, which significantly improves the amplification uniformity of MDA. However, skill barrier still exists for biological researchers to handle chip fabrication and droplet manipulation. Here, we present a novel MDA protocol, in-capillary MDA (icMDA), which significantly simplifies the manipulation and improves the uniformity of amplification by dispersing reagents in a long quasi-1D capillary tubing. We demonstrated that icMDA is able to accurately detect SNVs with higher efficiency and sensitivity. Moreover, this straightforward method employs neither customized instruments nor complicated operations, making it a ready-to-use approach for most laboratories.
منابع مشابه
Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification
Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage becaus...
متن کاملPicodroplet partitioned whole genome amplification of low biomass samples preserves genomic diversity for metagenomic analysis
BACKGROUND Whole genome amplification (WGA) is a challenging, key step in metagenomic studies of samples containing minute amounts of DNA, such as samples from low biomass environments. It is well known that multiple displacement amplification (MDA), the most commonly used WGA method for microbial samples, skews the genomic representation in the sample. We have combined MDA with droplet microfl...
متن کاملDecontamination of MDA Reagents for Single Cell Whole Genome Amplification
Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexa...
متن کاملWhole genome amplification of the rust Puccinia striiformis f. sp. tritici from single spores.
Rust fungi are obligate parasites and cannot be routinely cultured to obtain sufficient biomass for DNA extractions. Multiple displacement amplification (MDA) was demonstrated in this study for whole genome amplification from single spores of the rust fungus, Puccinia striiformis. The genomic DNA coverage and fidelity of this method was evaluated by PCR amplification and sequencing of two genet...
متن کاملQuantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA.
Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in...
متن کامل