Development of a novel microarray data analysis tool without normalization for genotyping degraded forensic DNA.

Since the arrest of the Golden State Killer in the US in April 2018, forensic geneticists have been increasingly interested in the investigative genetic genealogy (IGG) method. While this method has already been in practical use as a powerful tool for criminal investigation, we have yet to know well the limitations and potential risks. In this current study, we performed an evaluation study focusing on degraded DNA using the Affymetrix Genome-Wide Human SNP Array 6.0 platform (Thermo Fisher Scientific). We revealed one of the potential problems that occur during SNP genotype determination using a microarray-based platform. Our analysis results indicated that the SNP profiles derived from degraded DNA contained many false heterozygous SNPs. In addition, it was confirmed that the total amount of probe signal intensity on microarray chips derived from degraded DNA decreased significantly. Because the conventional analysis algorithm performs normalization during genotype determination, we concluded that noise signals could be genotype-called. To address this issue, we proposed a novel microarray data analysis method without normalization (nMAP). Although the nMAP algorithm resulted in a low call rate, it substantially improved genotyping accuracy. Finally, we confirmed the usefulness of the nMAP algorithm for kinship inferences. These findings and the nMAP algorithm will make a contribution to the advance of the IGG method.

Practical forensic use of kinship determination using high-density SNP profiling based on a microarray platform, focusing on low-quantity DNA.

Instead of traditional short tandem repeat (STR) profiling, the genetic genealogy method, which uses hundreds of thousands of single nucleotide polymorphisms (SNPs) spread across genome-wide, has emerged as a powerful kinship determination tool and recently attracted great attention in forensic genetics. In this study, we explored the tolerance and viability of kinship discrimination based on a high-density SNP profile for forensic DNA, especially focusing on low-quantity DNA. Using the Affymetrix Genome-Wide Human SNP Array 6.0 platform (Thermo Fisher Scientific), the influence of low-quantity DNA on SNP genotype determination was evaluated. The low-quantity DNA samples failed once every few samples, the generated SNP profile had low data quality. Our investigation revealed that the SNP profile with low data quality contained many genotyping errors in which the SNP genotype changed from homozygote to heterozygote. The kinship discrimination analysis using KING software was directly influenced by these genotyping errors, which was confirmed that some unrelated pairs were mis-specified as 4th-degree relatives. We confirmed that the false heterozygous SNPs resulted in an inflation of kinship coefficient and a decrease of non-shared allele between a tested pair. To eliminate the influence of these genotyping errors and acquire an accurate kinship discrimination result, we developed a novel method to select only the robust SNPs, which stably give the genotype determination with high accuracy even in SNP profiles with low data quality. The application of our novel method led to the improved results of kinship discrimination up to the same level as in the SNP profile with high data quality. In addition, this study demonstrated the advantage of kinship analysis using a high-density SNP profile in the forensic field. It is well known that likelihood ratio calculation based on autosomal STR profile, which is the most commonly applied approach, has difficulty in gaining true kinship analysis results, especially when the relationship between the tested two individuals is more biologically distant. We showed the kinship discrimination analysis with a high-density SNP profile is more suitable for the case without close relatives, using the real case data. Although further study with larger samples will be necessary, this study indicated that practical forensic use of kinship determination with a high-density SNP profile would bring benefits to the forensic field.

Genetic analyses of fancy rat-derived mutations.

To collect rat mutations and increase the value of the rat model system, we introduced fancy-derived mutations to the laboratory and carried out genetic analyses. Six fancy rats were shipped from a fancy rat colony in the USA and used as founders. After initial crosses with a laboratory strain, TM/Kyo or PVG/Seac, inbreeding started and 6 partially inbred lines, including 2 sublines, were produced as Kyoto Fancy Rat Stock (KFRS) strains. During inbreeding, we isolated 9 mutations: 5 coat colors, American mink (am), Black eye (Be), grey (g), Pearl (Pel), siamese (sia); 1 coat pattern, head spot (hs); 2 coat textures, Rex (Re), satin (sat); and an ear pinnae malformation, dumbo (dmbo). Genetic analyses mapped 7 mutations to particular regions of the rat chromosomes (Chr): am to Chr 1, sia to Chr 1, sat to Chr 3, Re to Chr 7, g to Chr 8, dmbo to Chr 14, and hs to Chr 15. Candidate gene analysis revealed that a missense mutation in the tyrosinase gene, Ser79Pro, was responsible for sia. From mutant phenotypes and mapping positions, it is likely that all mutations isolated in this study were unique to the fancy rat. These findings suggest that fancy rat colonies are a good source for collecting rat mutations. The fancy-derived mutations, made available to biomedical research in the current study, will increase the scientific value of laboratory rats.