Our software applies to human genomic MLPA probe design only but will be generalized in the future to deal with other genomes. In addition, H-MAPD is currently only for the design of MLPA copy number assays, but may be generalized in the future to deal with the design of MLPA assays for other applications, such as SNP detection and methylation changes. For multiple input sequences, the stuffer and bead tag modifications will be applied to the input sequences sequentially. For example, in bead-coupled MLPA, the first bead tag will be inserted into the first input sequence and so on.
The software takes into consideration most factors predicted to affect MLPA probe performance. However, there are likely to be unexpected factors that might cause problems. These factors include secondary structure of genomic DNA at the hybridization site, the presence of un-described SNP(s) at the hybridization site, sequence errors in the reference genome assembly, etc. The software performs sequence homology searches using BLAT due to its speed and excellent programming interface. Another popular sequence search tool is BLAST. Because they are implemented using different algorithms, BLAT and BLAST may not return identical results. The authors strongly recommend that users verify H-MAPD results with BLAST for independent validation.
Two platforms are available for MLPA assays. The electrophoresis-based platform requires the use of capillary electrophoresis systems which are available in most institutions; however long oligonucleotides (> 150 nt) in high quality are difficult to synthesize currently. The bead-coupled platform works with half probes (LPO/RPO) less than 100 nucleotides in length, but requires the use of a Luminex system. For the electrophoresis-based platform, probe length increase is achieved either by inserting stuffer sequences between the PCR primer and hybridizing sequences, or by extending the length of hybridizing sequences. By using stuffer sequences, hybridizing sequences have identical length and therefore have a more unified Tm. Since the hybridizing sequences are short, the use of stuffer provides the potential for detecting mutations/polymorphisms that are in close proximity to each other. However, designing stuffer sequences takes time. The stuffer sequences in additional file 3 have been verified in advance so that the union of default PCR primer (used by the commercial MRC-Holland MLPA kits) and stuffer sequences (default left primer + left stuffer; right stuffer + default right primer) are free of secondary structure or significant homology in the human genome. Some workers prefer extending hybridizing sequences to the use of stuffer sequences. However, longer hybridizing sequences are not suitable for short target regions and tend to result in more non-specific binding to the target genome. For the bead-coupled platform, the tag sequence can theoretically be incorporated in either the LPO or RPO. Currently, since anti-tags are linked at their 5' end to the commercially available FlexMAP beads, we recommend inserting the tag sequences in the RPO as illustrated in additional file 2. Thus, only the right PCR primer is in physical proximity to the bead, minimizing any steric hindrance between the PCR product and the bead. Another reason favouring inserting the tag in the RPO is that quite a few tag sequences start with adenosine (additional file 4). These tags will affect probe signal strength if inserted between the left primer and LHS. The commercial FlexMAP tag sequences were not designed specifically for MLPA assays, and some tag sequences, when attached to the default right primer, will form significant secondary structure on their own. For example, the tags corresponding to bead 062 and 071, when attached to the default right primer, have secondary structures that are significant: ΔG = -1.538 and -1.514 respectively (additional file 4). To avoid the use of these tags, the user can insert a short dummy sequence (for example, ACGT) in the input sequence corresponding to that tag (for example, when designing an assay with tags attached to the right primer, input sequence 62 should be input as a dummy sequence, so that tag 062 is consumed by the dummy sequence). Future development of H-MAPD should allow users to use their own stuffer sequences or bead tag sequences. H-MAPD allows users to specify custom PCR primers. However, users should be careful that the union of custom primers to the stuffer/tag sequences may result in secondary structures or significant homology to the human genome, even before specific hybridizing sequences are appended.
It is a challenge to design MLPA probes that can distinguish closely related sequences in the genome. For highly similar but non-identical sequences, H-MAPD will treat them as non-specific matches and is likely to fail the probe set. One can design MLPA probes for highly related sequences by allowing multiple perfect matches to amplify the common identical fragments. Of course the result represents multiple targets, and can not be used to distinguish the highly related but different sequences. A practical solution, as indicated in the MRC-Holland probe design guidelines , is to find the exact difference between the sequence of interest and its related sequences, and place the difference at the end of LPO or RPO. A branch of H-MAPD specifically designed for this purpose will be implemented in the future.