Validity of low copy number typing and applications to forensic science

Abstract

Low copy number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. There are a number of methodologies to increase sensitivity of detection to enable LCN typing. These approaches encompass modifications during the polymerase chain reaction (PCR) and/or post-PCR manipulations. Regardless of the manipulations, when processing a small number of starting templates during the PCR exaggerated stochastic sampling effects will occur. The result is that several phenomena can occur: a substantial imbalance of 2 alleles at a given heterozygous locus, allelic dropout, or increased stutter. With increased sensitivity of detection there is a concomitant increased risk of contamination. Recently, a commission reviewed LCN typing and found it to be "robust" and "fit for purpose." Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing. The findings of the commission seem inconsistent with the nature of LCN typing. While LCN typing is appropriate for identification of missing persons and human remains and for developing investigative leads, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. A more in-depth evaluation by the greater scientific community is warranted. The issues to consider include: training and education, evidence handling and collection procedures, the application or purpose for which the LCN result will be used, the reliability of current LCN methods, replicate analyses, interpretation and uncertainty, report writing, validation requirements, and alternate methodologies for better performance.

Figure 1

(A) With sufficient DNA, eg, 250 pg, faithful reproduction of the alleles can be generated. (B) Possible stochastic effects when a low copy number (LCN) sample containing approximately 36 pg of DNA is analyzed. The result can be heterozygote peak imbalance, allele drop-out (or a pseudo-homozygote profile), increased stutter, or combinations thereof. (C) The possible stochastic effects when a LCN sample containing approximately 36 pg of DNA is analyzed. Due to increased sensitivity of detection the risk of allele-drop in (and allele drop-out) is exacerbated.

Figure 3

There are a number of ways to increase sensitivity of detection for low copy number (LCN) typing. The one displayed here is post-polymerase chain reaction (post-PCR) purification using microcon dialysis to remove ions that compete with DNA during electrokinetic injection. A known 250 pg DNA sample was analyzed under standard operating profile conditions. The same sample was diluted to 7.8 pg and 2 replicates were independently amplified using the AmpFlSTR® Profiler® PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA). To increase sensitivity of detection, the 7.8 pg samples were subjected to post-PCR purification and a 4-fold increase of sample was used for analysis. The example shows the results for a subset of the full profile (D3S1358, vWA, and FGA loci). Typical stochastic effects are observed for the LCN samples.

Figure 2

A known DNA sample divided into 6 reactions, each containing 100 pg of DNA and independently amplified using the AmpFlSTR® Profiler Plus® ID PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA) and following the manufacturer’s recommended amplification conditions including 28 cycles for the polymerase chain reaction, but using a 40 relative fluorescence unit (RFU) detection threshold. The example shows the results for a subset of the full profile (D3S1358, vWA, and FGA loci). The top panel is a control sample run with sufficient DNA for conventional typing. The 6 panels below the control are the 100 pg replicates. This example demonstrates that replicates of pristine control samples containing 100 pg of DNA or less may not yield reproducible results. The problems associated with the amplification and interpretation of low copy number (LCN) samples are greatly compounded when evidentiary mixtures are analyzed. The lack of reproducibility will persist with methods employed to increase sensitivity of detection for LCN typing.