Shotgun metagenomics of biological stains using ultra-deep DNA sequencing

A detailed molecular analysis of blood or other biological stains at a crime scene is often hampered by the low quantity and quality of the extractable DNA. However, the determination of the origin and composition of a stain is in most cases a prerequisite for the final elucidation of a criminal case. Standard methodologies, e.g. amplification of DNA followed by microsatellite typing or mitochondrial DNA sequencing, are often not sensitive enough to result in sufficient and conclusive data. We have applied ultra-deep DNA sequencing using the 454 pyrosequencing technology on a whole genome amplified (WGA) environmental biological stain, which was analysed unsuccessfully with standard methodologies following WGA. With the combination of WGA and 454 pyrosequencing, however, we were able to generate 7242 single sequences with an average length of 195 bp. A total of 1,441,971 bp DNA 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, i.e. 4.2% bacterial DNA, 0.3% viral DNA, 2.7% fungal DNA, 10.3% mammalian repetitive DNA, 0.9% porcine DNA, 0.13% human DNA and 81.5% DNA of unknown origin. Our data demonstrate 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.