A low number of reads in digitalMLPA no-DNA reactions is usually not problematic. The number of unique (distinct) reads of any probe in no-DNA reactions is typically less than 1% of the number obtained in reactions with sample DNA. However, even these very low numbers of distinct reads can result in a considerable number of total reads for some probes. This does not significantly influence results in reactions with sample DNA, as the nonspecific reads will be outcompeted by the much larger number of amplicons derived from normal ligation events on sample DNA.

Background

The cause of the nonspecific distinct reads for some probes in no-DNA reactions is often the ligation of two probe oligos while they are (temporarily) annealed to another probe oligo. When these two probe oligos are from different probes, Coffalyser digitalMLPA automatically removes the reads from the results. However, when they are from the same probe, the reads will be assigned to that probe, and a nonspecific signal will be obtained.

We thoroughly test each probemix with all barcodes from barcode plates suitable for the probemix to ensure its reliability. Low background signals in no-DNA reactions are permitted if they do not lead to false results in reactions with sample DNA. Probes that give an elevated background signal in no-DNA reactions are marked with a warning in the Probe Information File (PIF).

However, it is important to note that small background signals in no-DNA reactions can also arise from:

1. Contamination of the NGS sequencer with amplicons from a previous run (carry-over). This has been described repeatedly for the MiSeq instrument (e.g. here). The presence of a significant number of reads from unused barcodes in the experiment can serve as an indicator of contamination with reads from a previous experiment. To minimise the chance of carry over, we recommend using different barcodes in subsequent NGS runs.
2. Contamination of the no-DNA reaction with human DNA. Contamination of reactions with sample DNA with more than 5% DNA of another individual is detected by Coffalyser digitalMLPA based on results of 39 SNP-specific probes with a high minor allele frequency in all populations.
3. Contamination of the no-DNA reaction with PCR amplicons from previous digitalMLPA experiments. When the contamination is derived from reactions with the same probemix, this will result in reads for (almost) all probes. When the contamination is derived from a reaction with a different digitalMLPA product, only a small number of probes, and all control probes, will show reads. Contamination with PCR amplicons from previous experiments will usually be apparent by the presence of substantial read numbers assigned to unused barcodes.

These contaminations typically generate a very small number of distinct reads for some, or (almost) all probes. When the number of distinct reads in no-DNA reactions is very low as compared to the median number of distinct reads in reactions with sample DNA, this will not influence the results.