Autosomal InDels for Identification
Development
This exercise will comprise two phases. The first phase will provide the participating laboratories a mix of primers needed to perform genotyping of 38 autosomal indels in four control samples. The method for genotyping is straightforward and similar to that performed for STRs comprising a single multiplex PCR followed by capillary electrophoresis (Pereira et al., 2009; Pereira & Gusmão, 2012).
Implementing the technique properly and the correct genotyping of samples from this first step will allow the lab to access the second phase of the exercise. In this second phase, it is recommended that each laboratory characterizes the reference population(s) of interest to construct a genetic database appropriate to its routine (minimum of 100 samples of a population).
For this exercise, an aliquot of the primer mix for a sufficient number of 500 reactions will be provided to the participating laboratories. Laboratories interested in characterizing a larger number of samples/populations (for inclusion in this study) may be sent an aliquot of extra primers upon request.
Laboratory work comprises the following steps:
1. Multiplex PCR. The 38 markers are amplified in a single PCR reaction using the primer mix provided. The primer mix is optimized to obtain balanced reaction products following the protocol indicated. The original protocol uses the PCR master mix "QIAGEN Multiplex PCR Kit."
2. Electrophoretic separation of amplified fragments. To be performed in a capillary sequencer such as "ABI Genetic Analyzer" (310, 3100, 3130, 3500, 3730 or equivalent variants). The original protocol uses POP7 as the separation polymer (alternatively, POP6 or POP4 may be used, always taking into account the consequent changes of electrophoretic mobility).
(Note: between steps 1 and 2, a further cleanup step of the PCR product may be carried out).
A properly processed sample should result in an electropherogram similar to that shown in the figure.
To the participating laboratories, an aliquot of 500 uL of PCR primer mix will be sent allowing the processing of approximately 500 reactions (mixture already prepared containing dye-labeled primer pairs, needed to the co-amplification of the 38 markers. Any other material or reagent required for completion of the study should be obtained by the participating laboratory.
For publication of the final results, there is a limit of one author per laboratory for a minimum of 100 individuals sampled per population. There may be a maximum of two authors accepted per participating laboratory when data corresponding to at least two populations is submitted, each comprising at least 100 samples.
For additional information or doubts regarding the reaction protocol and genetic profiling, please contact Rui Pereira (rpereira@ipatimup.pt). Template files of "panels" and "bins" to import into the analysis program (eg. GeneMapper), including support for mobility adjustment of each apparatus, can be requested to the same email address.
The genotypes obtained in the first step should be sent by January 31st, 2013. The results should be sent indicating the genotypic profile of the samples accompanied by the image of the electrophoregrams obtained, in pdf format.
The genotyping of the populations analyzed in the second stage should be reported until June 30th, 2013. The results must be submitted in excel format according to available template.
It is recommended to save the .fsa files and/or electrophoregrams of all runs performed as they may be needed to clarify questions that may arise.
Requirements for participants
The person responsible must be a GHEP member and have the membership fees up to date.
Payment of the participation fee of 150 euros on time (on-line with credit card or bank transfer according to http://www.gep-isfg.org/en/proficiency-testing/payment-procedures.html)
Deadlines
Deadline for registration: October 31, 2012
Deadline for payment: November 30, 2012
Deadline for submission of results: Step 1: January 31, 2013 Step 2: June 30, 2013
Contact
Rui Pereira (IPATIMUP, Porto, Portugal): rpereira@ipatimup.pt
Leonor Gusmao (IPATIMUP, Porto, Portugal): lgusmao@ipatimup.pt
References