Massively Parallel Sequencing for Forensic DNA Analysis

HiResToday’s blog is written by guest blogger, Douglas R. Storts, PhD, head of Research, Nucleic Acid Technologies, Promega Corporation.

Massively parallel sequencing (MPS), also called next generation sequencing (NGS), has the potential to alleviate some of the biggest challenges facing forensic laboratories, namely degraded DNA and samples containing DNA from multiple contributors. Unlike capillary electrophoresis, MPS genotyping methods do not require fluorescently-labeled oligonucleotides to distinguish amplification products of similar size. Furthermore, it is not necessary to design primers within a color channel to generate amplicons of different sizes to avoid allele overlap. Consequently, all the amplicons can be of a similar, small size (typically <275 base pairs). The small size of the amplicons is particularly advantageous when working with degraded DNA. Because the alleles are distinguished by the number of repeats and the DNA sequence, additional information can be derived from a sample. This can be especially important when genotyping mixtures. As previously demonstrated (1), this sequence variation can help distinguish stutter “peaks” from minor contributor alleles.

Because there is no reliance upon size and fluorescent label, significantly greater multiplexing is possible with MPS approaches. In addition to autosomal short tandem repeats (STRs), we can also sequence Y-STRs, single nucleotide polymorphisms (SNPs), and the mitochondrial DNA control region. The advantage to this approach is the forensic analyst does not need a priori knowledge whether a sample would benefit most from the different methods of genotyping.

Despite these major advantages, there are limitations to the near-term, broad deployment of current MPS technology into forensic laboratories. The limitations fall into four main categories:  Workflow, costs, performance with forensically-relevant samples, and community guidelines. Continue reading “Massively Parallel Sequencing for Forensic DNA Analysis”

“Fingerprinting” Your Cell Lines

Working in the laboratoryResearchers working with immortalized cell lines would readily agree when I state that it is almost impossible to look at cells under the microscope and identify them by name. There are phenotypic traits, however they do change with change in media composition, passage number and in response to growth factors. I remember the pretty arborizations my neuroblastoma cell line SH-SY5Y exhibited in response to nerve growth factor treatment. Thus physical appearance is not a distinguishing feature. Currently, in many labs, researchers typically use more than one cell line, and more than likely, share the same lab space to passage cells and the same incubator to grow the cells. In such scenarios, it is not difficult to imagine that cell lines might get mislabeled or cross-contaminated. For example HeLa cells, one of the fastest growing cell lines have been shown to invade and overtake other cell lines.

Misidentification of cell lines has deep and severe implications. A review of cell lines used to study esophageal adenocarcinoma found that a large number of the cell lines were actually derived from lung or gastric cancers. Unfortunately, by the time this error was discovered, data from these cell line studies were already being used for clinical trials and other advanced studies and publications. Moreover, the cell lines were being to screen and design and test specific cancer drugs which ended up in flawed clinical trials. Continue reading ““Fingerprinting” Your Cell Lines”