FindPrimers {EventPointer} | R Documentation

## FindPrimers

### Description

FindPrimers is the main function of the primers design option. The aim of this function is the design of PCR primers and TaqMan probes for detection and quantification of alternative splicing.

Depending on the assay we want to carry out the the algorithm will design the primers for a conventional PCR or the primers and TaqMan probes if we are performing a TaqMan assay.

In the case of a conventional PCR we will be able to detect the alternative splicing event. Besides, the algorithm gives as an output the length of the PCR bands that are going to appear. In the case of a TaqMan assay, we will not only detect but also quantify alternative splicing.

### Usage

    FindPrimers(
      SG,
      EventNum,
      Primer3Path,
      Dir,
      mygenomesequence,
      taqman = NA,
      nProbes = 1,
      nPrimerstwo = 3,
      ncommonForward = 3,
      ncommonReverse = 3,
      nExons = 5,
      nPrimers = 15,
      shortdistpenalty = 2000,
      maxLength = 1000,
      minsep = 100,
      wminsep = 200,
      valuethreePenalty = 1000,
      minexonlength = 25,
      wnpaths = 200,
      qualityfilter = 5000
    )

### Arguments

`SG` | Information of the graph of the gene where the selected event belongs. This information is avaible in the output of EventsGTFfromTranscriptomeGTF function.
`EventNum` | The "EventNum" variable can be found in the returned .txt file from the EventsGTFfromTranscriptomeGTF function in the column "EventNumber" or in the output of EventPointer_RNASeq_TranRef, the number after the "_" character of the 'Event_ID'.
`Primer3Path` | Complete path where primer3_core.exe is placed.
`Dir` | Complete path where primer3web_v4_0_0_default_settings.txt file and primer3_config directory are stored.
`mygenomesequence` | genome sequence of reference
`taqman` | TRUE if you want to get probes and primers for taqman. FALSE if you want to get primers for conventional PCR.
`nProbes` | Number of probes for Taqman experiments. By default 1.
`nPrimerstwo` | Number of potential exon locations for primers using two primers (one forward and one reverse). By default 3.
`ncommonForward` | Number of potential exon locations for primers using one primer in forward and two in reverse. By default 3.
`ncommonReverse` | Number of potential exon locations for primers using two primer in forward and one in reverse. By default 3.
`nExons` | Number of combinations of ways to place primers in exons to interrogate an event after sorting. By default 5.
`nPrimers` | Once the exons are selected, number of primers combination sequences to search within the whole set of potential sequences. By default 5.
`shortdistpenalty` | Penalty for short exons following an exponential funciton(A * exp(-dist * shortdistpenalty)). By defautl 2000.
`maxLength` | Max length of exons that are between primers and for paths once we have calculated the sequence. By default 1000.
`minsep` | Distance from which it is penalized primers for being too close By default 100.
`wminsep` | Weigh of the penalization to primers for being too close By default 200.
`valuethreePenalty` | penalization for cases that need three primers instead of 2. By default 1000.
`minexonlength` | Minimum length that a exon has to have to be able to contain a primer. By default 25.
`wnpaths` | Penalty for each existing path By default 200.
`qualityfilter` | Results will show as maximum 3 combinations with a punctuation higher than qualityfilter By default 5000.

### Value

The output of the function is a 'data.frame' whose columns are:

For1Seq: Sequence of the first forward primer.

For2Seq: Sequence of the second forward primer in case it is needed.

Rev1Seq: Sequence of the first reverse primer.

Rev2Seq: Sequence of the second reverse primer in case it is needed.

For1Exon: Name of the exon of the first forward primer.

For2Exon: Name of the exon of the second forward primer in case it is needed.

Rev1Exon: Name of the exon of the first reverse primer.

Rev2Exon: Name of the exon of the second reverse primer in case it is needed.

FINALvalue: Final punctuation for that combination of exons and sequences. The lower it is this score, the better it is the combination.

DistPath1: Distances of the bands, in base pairs, that interrogate Path1 when we perform the conventional PCR experiment.

DistPath2: Distances of the bands, in base pairs, that interrogate Path2 'when we perform the conventional PCR experiment.

DistNoPath: Distances of the bands, in base pairs, that they do not interrogate any of the two paths when we perform the conventional PCR experiment.

SeqProbeRef: Sequence of the TaqMan probe placed in the Reference.

SeqProbeP1: Sequence of the TaqMan probe placed in the Path1.

SeqProbeP2: Sequence of the TaqMan probe placed in the Path2.

### Examples

    ## Not run:

    data("EventXtrans")
    #From the output of EventsGTFfromTranscriptomeGTF we take the splicing graph information
    SG_list <- EventXtrans$SG_List
    #SG_list contains the information of the splicing graphs for each gene

    #Let's supone we want to design primers for the event 1 of the gene ENSG00000254709.7

    #We take the splicing graph information of the required gene
    SG <- SG_list$ENSG00000254709.7

    #We point the event number
    EventNum <- 1

    #Define rest of variables:
    Primer3Path <- Sys.which("primer3_core")
    Dir <- "C:\\PROGRA~2\\primer3\\"

    MyPrimers <- FindPrimers(SG = SG,
                             EventNum = EventNum,
                             Primer3Path = Primer3Path,
                             Dir = Dir,
                             mygenomesequence = BSgenome.Hsapiens.UCSC.hg38::Hsapiens,
                             taqman = 1,
                             nProbes=1,
                             nPrimerstwo=4,
                             ncommonForward=4,
                             ncommonReverse=4,
                             nExons=10,
                             nPrimers =5,
                             maxLength = 1200)

    ## End(Not run)

[Package EventPointer version 3.0.0 Index]