Single Cell Genome Amplification

Background whole genome amplification wga is currently a prerequisite for single cell whole genome or exome sequencing.

Single cell genome amplification.

Methodology and applications annu rev genomics hum genet. Whole genome amplification from a single cell is now possible with our optimized genomeplex single cell whole genome amplification kit the single cell procedure differs very little from the previously described genomeplex system but for three procedural changes. Reagents required for mda reactions include. Single cell genomiphi has been developed with this in mind.

Wga is achieved by pcr amplification of the library molecules using universal. Here we report an improved single cell wga method linear amplification via trans. Depending on the method used the rate of artifact formation allelic dropout and sequence coverage over the genome may differ significantly. Epub 2015 jun 11.

Genomeplex single cell whole genome amplification kit utilizes a proprietary technology based on random fragmentation of genomic dna and conversion of the resulting small fragments to pcr amplifiable library molecules flanked by universal priming sites. 1 the kit includes a robust optimized cell lysis protocol that is incorporated into the. In contrast a deep analysis of the genome currently requires a few hundred nanograms up to micrograms of genomic dna for library formation necessary for ngs sequencing or labeling protocols e g microarrays. We present a survey of single cell whole genome amplification wga methods including degenerate oligonucleotide primed polymerase chain reaction dop pcr multiple displacement amplification mda and multiple annealing and looping based amplification cycles malbac.

Results the largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth. Single cell genomics is important for biology and medicine. Single cell genomiphi dna amplification kit has been optimized to wholly amplify genomic dna from as little as a single cell generating micrograms of high quality dna for use in downstream applications. E coli as reference genome is an ideal model system to identify amplification bias introduced via technology used.

A list of more than 100 different single cell omics methods have been published. At very low levels of input dna successful amplification relies on reagent quality. However current whole genome amplification wga methods are limited by low accuracy of copy number variation cnv detection and low amplification fidelity. Authors lei huang 1 fei ma alec chapman sijia lu xiaoliang sunney xie.

Consequently accurate whole genome amplification wga of single cell dna is required for reliable genetic analysis e g ngs. Random primers and dna polymerase from bacteriophage phi29. Affiliation 1 biodynamic optical. 1 pg of e coli dna has been amplified with either the repli g single cell kit or a kit based on malbac technology library prep and miseq sequencing similar data analysis on clc workbench.