In this exercise the participating laboratories will be supplied with a primer mix needed to perform genotyping of all samples included in the 2014 Intercomparison Exercise. The primer mix includes all primers necessary for PCR amplification of six regions of the mitochondrial DNA (mtDNA) rich in insertion/deletion polymorphisms (indels) originating profiles of unique sizes for the following mammalian species: human (Homo sapiens) cat (Felis catus), dog (Canis familiaris), cow (Bos taurus), goat (Capra hircus), sheep (Ovis aries), pig (Sus scrofa), rabbit (Oryctolagus cuniculus), horse (Equus caballus) and mouse (Mus musculus). However, unique profiles may be obtained for other species as discussed in reference Pereira et al., 2010.
The genotyping methodology is straightforward and similar to that employed with STRs involving a single multiplex PCR followed by capillary electrophoresis.
Laboratory work comprises the following steps:
1. Multiplex PCR. The 6 markers (named SPID) 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).
The resulting profiles of properly processed samples should result in electrophoregrams similar to those shown in the picture:
To the participating laboratories, an aliquot of 100 µL of PCR primer mix will be sent allowing the processing of approximately 100 reactions. The mixture contains fluorescently labelled primer pairs (6-FAM) needed to co-amplify the 6 markers. Any other materials or reagents required for the study must be obtained by the participating laboratory.
For additional information or clarification of doubts concerning the PCR protocol and genetic profiling please contact Filipe Pereira (via e-mail email@example.com). 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.
After genotyping, sample classification in terms of determining the species can be achieved by SPInDel platform, available in http://www.portugene.com/SPInDel/SPInDel_web.html. The program includes a database containing the reference sequences of mtDNA and the numerical profiles obtained with SPID markers.
The results should be sent until one month after the deadline for submission of the GHEP-ISFG 2014 Intercomparison Exercise. The results should be sent indicating the profile obtained (in format that will be disclosed later), accompanied by the respective electrophoregrams in pdf format.
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 participants must be GHEP members and have the membership fees up to date.
• Payment of the participation fee of 50 euros on time (online with credit card or bank transfer, according to http://www.gep-isfg.org/en/proficiency-testing/payment-procedures.html)
Deadline for registration: December 31st, 2013
Deadline for payment: February 7th, 2014
Deadline for submission of results: one month after the deadline established for the GHEP Intercomparison Exercise 2014.
Pereira F, Carneiro J, Matthiesen R, van Asch B, Pinto N, Gusmão L, Amorim A. Identification of species by multiplex analysis of variable-length sequences. Nucleic Acids Res. 2010 Dec;38(22):e203.
Carneiro J, Pereira F, Amorim A. SPInDel: a multifunctional workbench for species identification using insertion/deletion variants. Mol Ecol Resour. 2012 12(6):1190-5.