So you've sent your swab back to us. What happens next? Read on to see the processes your sample goes through to get your set of numbers that allows you to compare yourself with your relatives and thousands others worldwide.

Laboratory Preparation
Workflow is always unidirectional from DNA Extraction through to Detection which ensures that cross-contamination of DNA samples doesn't occur. This includes specimens, reagents, and paperwork.

Every laboratory work surface is disinfected with 10% bleach in water followed by a 100% ethanol rinse at least once per day. This removes any traces of contamination from the surface. General protocols require that all surfaces and equipment are cleaned at least once per day as well as before and after any operation or process setup.

To assure that there are undetectable levels of contamination, at least once per week two separate surfaces in the Extraction or PCR Setup areas are wipe tested to check for the presence of amplified DNA.

DNA Extraction

When a sample comes in, the bar-coded sample number is utilised to allow tracking of the sample at every stage. Each test is run in duplicate and the results married up at the end to ensure consistent results.

Before we can test the DNA, we have to extract it from the chromosomes from within the cells. We do this using a series of extractions.


Most extractions (i.e., swab specimens) are performed on our QIAGEN BioRobot 8000, which can extract 4 plates of 96 samples and controls (a total of 384 samples) at one time, simultaneously. Each extraction batch includes quality controls to monitor for, and detect possibilities of contamination. This ensures reagent quality and the specificity of the test.

All reagents for PCR are prepared in a dedicated dead air box, under low light conditions, and on ice to ensure contamination free master mix and optimum sensitivity for all fluorescent labels.

Extracted DNA, master mix, and controls are automatically combined and loaded onto 384 well plates.

Amplification

After PCR setup, the plates are transferred into the Amplification Laboratory through a dedicated one way pass-through box on the wall of the laboratory. This pass through box helps to minimize contamination and maintain unidirectional work flow into the amplification area.

The laboratory utilizes several ABI 9700 thermo cyclers. These are all networked into a central computer that serves the specific amplification protocols dependent on the test type and markers being amplified. Our Laboratory Information System (LIMS) records and tracks which machine and protocol is run for each plate.

Detection

After amplification, the amplified DNA is combined with formamide and size standard on our second Biomek FX robot. Formamide is a chemical that denatures the DNA amplicon and facilitates detection and electrophoresis. The size standard is called the 'Ladder' which ensures that each allele is correctly located in the associated bin for its specific fragment size.

The plate is quickly heated to 96 ºC for several minutes to facilitate the denaturation and then placed in the freezer for several minutes to 'freeze' in the denatured DNA.

The plate is then loaded onto any of our several ABI detection machines. We use a combination of machines for different purposes. We use an ABI 3100 for basic research and panel development. We use our ABI 3700's for high-throughput sequencing and finally we use our several ABI 3730's for high-throughput fragment analysis work.

Once the plate has been run through analysis it is placed in the Detection refrigerator for short term storage to ensure that the run passes all Quality Control checks.

Analysis

Our laboratory tracking system automatically uploads the data files from the detection instruments and launches GeneScan and GenoTyper software programs. These programs are used to assess the quality of each run and sample as well as to assign the specific alleles to each fragment analyzed.

Each sample in each batch run through Detection is analyzed and called by two independent analysts. Their calls are compared by the laboratory information system and any discrepancies are brought out for resolution by our lead analyst.

Since each swab specimen is extracted in duplicate, there are two distinct batches for each sample that comes off the detection instruments. This results in 4 separate readings of each marker for each person we test. If any discrepancy results then the sample may be recollected or the third swab extracted.

Quality Control

The following controls are used on every batch and plate processed, and is reviewed at the Analysis step:

- Negative Extraction. This is basically a check to ensure that the extraction reagents are clean and that no amplification occurs. If any peaks come up in this control the whole batch fails and is re-extracted.

- PCR Negative Control. Like the extraction negative control we ensure that the reagents and master mix for PCR are clean and do not give rise to spurious peaks.

- PCR Positive Control(s). One or more controls are added to each plate to ensure that the correct calls are being made for each plate. Positive controls use a combination of NIST SRM, CEPH DNA, and employee DNA samples. These controls allow for us to ensure that each plate runs correctly, that it gives the correct results without shifting data, and allows for an assessment of contamination control.

- DNA Ladders. Each sample's alleles line up within specified bins defined by the size standard or ladder each was processed with. A QC check is performed to ensure that each allele is within the expected bin location.

- Data matching between analysts and batches. The analysts ensure consistency between the two batches and the two independent reviews. This makes a total of 4 unique readings and reviews of each person's genotype.


Reporting

The lead analyst prints out the final report for the individual being reported. A clerical/administrative review of the case file and report is conducted to ensure that all is in order and that no typographic errors have been entered into the laboratory tracking system.

The final report is then reviewed by either the Laboratory Supervisor, Laboratory Manager, or the Laboratory Director, depending on the type of case.

Once all reviews are conducted the report is signed by the final reviewer(s) and uploaded to Oracle and released.