Improved DOP-PCR (iDOP-PCR): A robust and simple WGA method for efficient amplification of low copy number genomic DNA

Abstract

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

Current status of spent embryo media research for preimplantation genetic testing

“…PCR-based amplification using a single degenerate oligonucleotide primer (DOP-PCR) was among the first WGA methods employed to successfully detect aneuploidy using aCGH [26,27]. Relatively low coverage of the genome and high incidence of allele dropout (ADO) renders DOP-PCR inferior to more modern WGA techniques [28]. Methods using a combination of displacement pre-amplification and PCR-amplification such as the PicoPlex/SurePlex WGA system are widely used in PGT-A today and have been reported to cover~30% of the genome [25].…”

Algorithms for automated detection of hook effect-bearing amplification curves

“…1, Supplement Table 1). The raw data were compiled from the boggy data set [12] , the testdat data set [11] , the C127EGHP data set [13] , a whole genome amplification experiment [14] and from an in-house BRCA1 gene quantification experiment. The raw data were rated by the humanrater() function either as hook effect-bearing (“y”) or not (“n”), as described in the Supplementary Information of [13] .…”

Single-cell sequencing and its applications in bladder cancer

“…Recently, alternative WGA technologies having degenerate oligonucleotide-primed-PCR (Ref. 21), multiple displacement amplification ( Refs 21,22) and multiple annealing and loopingbased amplification cycles (MALBAC) (Ref. 23).…”