Genotype data for 768 STR markers were obtained for 15 MDA–DNAs and paired un-amplified genomic DNA (hereafter ‘gDNA’). Average genotyping call rate for the MDAs was 95.0% (91.4–98.2%), lower than the 96.5% (93.2–100%) achieved for the gDNA (). To further investigate genotyping failures, we categorised each no-call by the genotype of the gDNA. The MDA samples were found to have comparable failure rates for both heterozygous (5.4% of all heterozygote calls) and homozygous genotypes (4.1% of all homozygote calls). The MZ genomic controls showed heterozygote and homozygote failures of 3.5% and 2.6%, respectively. Thus, although there was a net increase of genotyping failure in the MDAs, we did not observe heterozygous genotypes to be more prone to failure than homozygotes.
For called genotypes in the MDA–DNA and paired gDNA, a genotype concordance rate of 97.8% (97.2–98.4%) was observed (). A genotype concordance of 99.2% (98.0–99.9%) was achieved for the 16 MZ gDNAs (). As MZ co-twins should have identical genotypes, we considered the discordance rate, 0.8% (1–0.992), to be an approximation of the error of the genotyping process, and indeed it was in good agreement with the 0.7% error rate reported by Marshfield Clinic Mammalian Genotyping Service (13
). If we assume a 0.8% base-line error rate, the overall increase in genotype discrepancies in the MDA samples was ~1.4%. We categorised discrepancies according to the observed change in assigned STR allele number, comparing the MZ2 to MZ1 samples, and the MDA to gDNA. Discrepancies were on average 2.9 times more common in the MDAs than in the MZ controls. Heterozygote to homozygote genotype transitions (loss of heterozygosity, LOH) were more common in the MDAs at 3.8 times the level observed in MZ pairs. Discrepancies involving one or both of the assigned allele values were found to occur at comparable rates for an allele size increase (2.2 times) versus a decrease (2.4 times). In summary, we observe a small increase in LOH in the MDA samples relative to other forms of discrepancy. Our data also indicates no marked tendency in the MDAs towards larger or smaller alleles.
To evaluate whether pooling of replicate MDA reactions prior to genotyping may even out stochastic variations in WGA efficacy as suggested by others (9
), we amplified five DNAs (a subset of the 15 DNAs above) in three independent replicate MDA reactions, which were then pooled for genotyping. Mean call rate for the pooled samples was 96.2% (94.9–97.5%) slightly improved over 95.8% (92.3–98.2%) for the five single-reaction MDAs from the same seed DNAs, and comparable to 96.1% (93.2–98.8%) for gDNA. MDA concordance with gDNA genotype showed a small but significant improvement in the pooled samples (98.3%) compared to single MDA (97.8%).
Next we assessed the individual genotyping failure rate and gDNA genotype concordance for each STR marker. We identified five STRs (ATA4E02, MFD427-AAAT028, ATT077P, GATA63C02, TAA005) that appear to be strongly prone to either failure and/or higher genotyping discrepancies. For these five markers, mean combined failure/discordance rate was 90.7% for the MDA–DNAs, and 88.0% for the triplicate-pooled MDAs. In contrast, we typically observe zero or no more than one genotyping failure (>10% failure/discordance rate) in the paired gDNA or MZ twins. Another 29 STRs were found to have a combined failure/discordance rate of ≥40% in both or either the 15 single MDAs and five pooled MDAs. These 34 STRs, 4.4% of all STRs in the genome-wide scan, account for 30.5% of all failures/discordance in the 15 single MDAs, and 38.0% in the five triplicate-pooled MDAs. In contrast, the 34 STRs account for only 4.8% of total anomalies/failures in our MZ controls. Repetitive genomic sequence, such as the centromere and telomere are known to be poorly represented in MDA–DNA (1
), so we considered the chromosomal position for each of the 34 STRs. They were found to be widely distributed across 17 chromosomes, with only two being the most-telomeric marker, and one was close to the centromere. Three of the five worst markers were close to a chromosome end but in no case were they the most-telomeric marker for that chromosome, suggesting other factors are important. Four of these five STRs had AAT or AAAT repeat motifs and the fifth was GATA. Of the 34 poor markers 14 (41.2%) had AAT or AAAT repeats which was higher than the average fraction (25.3%) of the genome set, just reaching significance (P
= 0.046, Fisher's Exact Test), whereas, there was no obvious enrichment for the GATA type (52.9% versus 61.8%). Thus, this type of repeat (exclusively AT) may be a factor in poor MDA replication but nevertheless is not usefully predictive of which STRs will be problematic. These same markers did not have failure rates or error rates on gDNA detectably different from the rest of the marker set as analysed from the full data set of the 1391 DNAs in the same experiment. When we removed the 34 problematic STRs from analysis, overall call rate for the MDA samples was very similar to that of gDNA, and genotype concordance was found to improve by 0.6% in the 15 single-reaction MDAs, and 0.4% in the five triplicate-pooled MDAs.