Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out.

Estimating an individual’s age can be relevant in several areas primarily related to the clinical and forensic fields. In the latter, estimation of an individual’s chronological age from biological material left by the perpetrator at a crime scene may provide helpful information for police investigation. Estimation of age is also beneficial in immigration cases, where age can affect the person’s protection status under the law, or in disaster victim identification to narrow the list of potential missing persons. In the last decade, research has focused on establishing new approaches for age prediction in the forensic field. From the first forensic age estimations based on morphological inspections of macroscopic changes in bone and teeth, the focus has shifted to molecular methods for age estimation. These methods allow the use of samples from human biological material that does not contain morphological age features and can, in theory, be investigated in traces containing only small amounts of biological material. Molecular methods involving DNA analyses are the primary choice and estimation of DNA methylation levels at specific sites in the genome is the most promising tool. This review aims to provide an overview of the status of forensic age prediction using molecular methods, with particular focus in DNA methylation. The frequent challenges that impact forensic age prediction model development will be addressed, together with the importance of validation efforts within the forensic community.

The Infinium MethylationEPIC BeadChip (EPIC) is a reliable method for measuring the DNA methylation of more than 850,000 CpG positions. In clinical and forensic settings, it is critical to be able to work with low DNA amounts without risking reduced reproducibility. We evaluated the EPIC for a range of DNA amounts using two-fold serial dilutions investigated on two different days. While the β-value distributions were generally unaffected by decreasing DNA amounts, the median squared Pearson’s correlation coefficient (R²) of between-days β-value comparisons decreased from 0.994 (500 ng DNA) to 0.957 (16 ng DNA). The median standard deviation of the β-values was 0.005 and up to 0.017 (median of medians: 0.014) for β-values around 0.6–0.7. With decreasing amounts of DNA from 500 ng to 16 ng, the percentage of probes with standard deviations ≤ 0.1 decreased from 99.9% to 99.4%. This study showed that high reproducibility results are obtained with DNA amounts in the range 125–500 ng DNA, while DNA amounts equal to 63 ng or below gave less reproducible results.

Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the ...