FindPrimers: FindPrimers

In jpromeror/EventPointer: An effective identification of alternative splicing events using junction arrays and RNA-Seq data

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 EventDetection_transcriptome function.
EventNum	The "EventNum" variable can be found in the returned .txt file from the EventDetection_transcriptome 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)

jpromeror/EventPointer documentation built on May 17, 2023, 10:29 p.m.