A new ultradeep LT (low template) DNA profiling approach based on an emulsion-based clonal amplification of an STRS multiplex PCR product followed by massive pyrosequencing

Due to an insufficient quantity and quality of the template DNA and often to the presence of PCR inhibitory compounds, frequently, evidentiary items collected at the crime scene do not allow to obtain complete or even partial DNA interpretable profiles using standard forensic STRs DNA analyses. This has led to introduce several modifications to the traditional STRs genotyping methodologies known as LT DNA profiling which often allow to recover all the genetic information even from a trace containing DNA at or beneath the stochastic threshold. Nonetheless LT DNA profiling suffers of several disadvantages known as stochastic sampling effects (exaggerated stutter, relevant peak height imbalance, allelic drop-out and allelic drop-in). In an attempt to overcome these drawbacks and to improve the sample DNA limit of detection of the new STRs multiplexes kits we developed a new approach involving a first step of multiplex PCR amplification by the new generation kits and an emulsion-based clonal amplification of the PCR products followed by a massively parallel pyrosequencing by the FLX-Titanium Genome Sequencer (Roche/454 Life Sciences). This new approach was conducted over several LT DNA samples (both pure DNA and DNA deriving from saliva and blood samples and real traces) at various dilution factors (down to 6 pg) and in comparison with the conventional new generation multiplexes. It revealed capable of genotyping each sample providing in about the 90% of samples full interpretable STRs DNA profiles quantitatively giving, for each sample, the complete STRs alleles population enclosed in a PCR product.