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.

Application of single cell genomics to focal epilepsies: A call to action

“…These methods that rely on non‐linear exponential amplification are degenerate oligonucleotide‐primed PCR (DOP‐PCR) (155) and multiple displacement amplification (MDA) (156). DOP‐PCR is fast, uniform, and readily accessible through popular commercial products such as GenomePlex (Sigma‐Aldrich) (157), but it has low coverage of the genome and is error prone (158). MDA is another popular and commercially available strategy for scWGA (159) that offers high coverage of the genome and low error rate, but it lacks uniformity (15).…”

HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling

“…Advances in single-cell DNA sequencing (scDNA-seq) technologies have enabled exploring new dimensions of genomic diversity that are not accessible by bulk sequencing. scDNA-seq methods have been instrumental in the fields of somatic mutation and mosaicism [1][2][3][4][5][6], organ development [7][8][9][10][11], germ cell mutation and fertility [12][13][14][15][16], epigenetic regulation and genome organisation [17][18][19][20][21][22][23] and cancer research [1,2,[24][25][26][27]. Established protocols conduct targeted sequencing, i.e.…”