This paper reports on a method of analyzing biological stains using ultra-deep DNA sequencing, which enables the analysis of trace samples of low quality (less than 5 pg g DNA, fragment sizes less than 100 bp) and an unbiased species identification.
The case in which this method was used involved the analysis of a blood stain of unknown origin and age present on the concrete floor of a storage building near a farm. The stain had been identified at a crime scene by a tracking dog and secured by the police. The authors applied ultra-deep DNA sequencing using the 454 pyrosequencing technology on the whole genome amplified (WGA) environmental biological stain after an unsuccessful analysis with standard methodologies following WGA. With the combination of WGA and 454 pyrosequencing, the authors were able to generate 7242 single sequences with an average length of 195 bp. A total of 1,441,971 bp sequences were generated and compared with public DNA sequence databases. Using RepeatMasker and basic logical alignment search tool (BLAST) searches against known microbial and mammalian genomes, it was possible to determine the metagenomic composition of the stain. Identifiable DNA composed 18.5 percent of the stain, and 81.5 percent of the stain had DNA of unknown origin. The identifiable composition consisted of varying percentages of bacterial, viral, fungal, mammalian repetitive, porcine, and human (0.13 percent) DNA. The data show that 454 pyrosequencing has the potential to become a powerful tool not only in basic research but also in the metagenomic analysis of biological trace materials for forensic genetics. The materials and methods used are described. 2 table and 34 references
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