What's behind the N4 DNA error?

PBB Data and Genetics Manager Megan Ellett says some PBB Neogen DNA clients have been receiving N4 errors following diagnostics of their SNP tested animals and she’s outlined how breeders can best reduce that result.

An N4 error means that the sample submitted had insufficient DNA material for an appropriate SNP.

Megan said the main reasons for an “N4” result are generally insufficient sample material or a contaminated sample.

There are two key steps to helping reduce the chances of returning an N4 error when DNA testing.

Those steps are choosing to test at a high level, such as the 100K SNP test and taking additional care when harvesting a DNA sample.

Analysis on the N4 results has found that samples with less than a 97% call rate (that means the percentage of SNP markers that could be taken from the DNA sample) will not always pass the AGBU quality control measures for inclusion in evaluations.

Using the higher-level testing, such as 100K is a strong step towards helping eliminate an N4 result, as it means an improved chance of reaching the required 97% call rate needed for AGBUs genomic evaluation.

Megan says DNA sample quality has a significant role to play in acceptable call rates. A lower call rate typically means poorer DNA quality. The simplest option when genotypes are flagged with an N4 error is resampling (by collecting a new sample).

“The easiest way to ensure less N4 errors is to submit quality DNA samples, that means taking particular care when collecting samples,” says Megan.

When using the Allflex TSU system it’s vital that the ear punch is sitting in the buffer liquid in the vile as this helps it retain viability.

TSUs should be kept at room temperature or in the fridge, they should not be placed in the freezer. Allflex recommends DNA testing occurs within 12 months of sample collection.

She also urges all breeders to ensure the tissue sample makes it into the vile and isn’t trapped in the TSU cap accidentally.

“There also shouldn’t be any hair sticking out of the tube as that means the sample isn’t sealed correctly, allowing the buffer to leak and the sample to dry out.”

When it comes to hair samples, she says it’s best practice to pluck the hair sample from the animal and not cut it.

“DNA is extracted from a hair sample via the root or bulb at the end of the hair follicle.”

To ensure a result can be obtained and the sample reaches an acceptable call rate, more than 30 hairs with roots attached need to be collected. The ideal place to take a sample is the tail switch. The more hair follicles supplied the better. A Neogen approved hair card should be used (available from the PBB office).

Samples can be cross-contaminated if they get wet or dirty. Contamination can also be caused from dyes such as those found in animal markers, animal health treatments and cleaning agents etc. Heat exposure can also lead to degradation of the sample if stored in direct sunlight or a hot position.

The correct storage of a sample is critical – see attached storage tip sheets.

In cattle, DNA can cross the placenta, which can mean mixed genotypes in non-identical/fraternal twins – otherwise known as blood chimeras.

“A blood chimera will generally fail it’s DNA quality control and have lower call rates,” says Megan.

Identical twins have identical DNA, so that means going forward any DNA sample that fails to reach the 97% call rate threshold will first be identified to see if it’s a twin. If that’s the case, a resample using hair should be submitted as it’s been found this type of sample results in higher percentage call rates for identical twins.

Key Points

  • There are ways to prevent N4 errors.
  • Follow the correct sampling process to ensure good quality samples.
  • Test at highest level possible e.g. 100K.
  • Test identical twins using hair sample.