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”

Do You #Authenticate?

authenticate_logoOn April 15, 2015 Nature announced a new policy around authentication of cell lines used in research studies that are published in its journals (1). Beginning in May 2015 they are asking all authors to confirm that they are not working on cells known to have been misidentified or cross-contaminated and to provide details about the source and testing of their cell lines.

The problem of cell line misidentification has been well documented in the literature with issues being reported with hematopoietic cell lines in 1999 (2) and a lymphoma cell line in 2001 (3). In 2006, one study suggested that 15–20% cells used in experiments have been misidentified or cross contaminated (4). And, in her book The Immortal Life of Henrietta Lacks, published in 2010, science writer Rebecca Skloot, notes that concern about cell line contamination dates back to 1958 (5). Promega has written about this problem and the power of STR analysis to assist you in assuring that your cell lines are what they should be (6–9). In fact, as John Masters, Professor of experimental pathology at University College, London points, out, there really is no excuse for the continuing problem of cell line contamination:

“For nearly 50 years, people have been using falsely identified cells totally unnecessarily because they haven’t checked.” (10)

Know what your cells are. #authenticate
Know what your cells are. #authenticate
The problem of cell line misidentification and contamination is not a new problem, and the calls for the scientific community to take extra care in understanding the identity of the cells that they are working with are not new either. Nature journals are not the first journals to take a stand to require authors to authenticate their cell lines. Journals including  International Journal of Cancer , In Vitro Cellular and Developmental Biology and Cell Biochemistry and Biophysics previously put policies in place around this issue (11,12), and in 2012 a new standard (ASN-0002) was officially published by the American National Standards Institute regarding human cell line authentication using profiles generated from STRs (11) . Based on the work of the ASN-0002 work group, the International Cell Line Authentication Committee was formed to promote awareness and authentication testing worldwide (13), including creating a publicly available database of misidentified cell lines.  However, as more and more high-profile cancer studies are retracted because of cell line issues (14,15), it has become apparent standards for cell line culture and authentication will need to become common place in life science research. Continue reading “Do You #Authenticate?”

“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”