Efficient sequencing and analysis of degraded and trace DNA samples using a novel targeted ligation-free method

Capturing information from trace and degraded DNA samples remains one of the most significant challenges for the field of DNA forensics. These samples generally contain DNA in fragments that are too small for traditional PCR and are thus not amenable to CODIS profiling; furthermore, they may not even contain complete copies of the donor’s genome.

High-throughput sequencing has the potential to recover information from these samples, as even small fragments can contain single nucleotide polymorphisms (SNPs) useful for identification, predicting visible characteristics such as ancestry and hair/eye color, and generating investigative leads.

Building on their past work, the researchers propose to develop a novel targeted enrichment strategy that, coupled with custom informatics methods, will generate investigative leads from highly-degraded forensic samples. This sample-to-report pipeline will consist of the following steps:

1) DNA extraction using a protocol optimized to retain small fragments;

2) Application of a novel ligation-free sequencing method for highly-degraded samples, targeted to a pre-selected panel of forensically relevant SNPs; and

3) Use of custom informatics methods to generate a report that includes sex, autosomal ancestry, maternal and paternal lineage, select phenotypic markers, and match probabilities with confidence levels.

The researchers targeted primer extension-based sequencing method involves the use of a single primer binding near the SNP of interest; this approach bypasses the need for two primer binding sites in a fragment (e.g., in PCR), enabling the inclusion of very small (<50 base pair) fragments. Furthermore, sequencing adapters are added without the need for ligation, which is known to be highly inefficient and results in sample loss.

This pipeline can be applied to extract information from samples for which CODIS genotyping failed, and can also provide investigative leads for cases in which no match is found in the CODIS database.

This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14).

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