1.1 Input data

Dataset required for haplotype statistic, visualization and phenotype association and the import function were listed in Table 1.

The genotype dataset is essential for haplotype identification and could be supplied in VCF, FASTA, P.link, HAPMAP and table format. The annotation were used for variants filtration and prepare schematic diagram.

Detailed information of individuals include phenotype data, group/category information and geo-coordinates. The phenotype data was used for comparison between different haplotypes. The group /category information was used for pie plot with haplotype network (eg. the second column in Table 4). And the geo-coordinates only used for demonstration of geographical distribution and include two columns: longitude and latitude (eg. the third and fourth column in Table 4).

Table 1: The required format of dataset and import functions for geneHapR

Table 2 is an example of genotypic data in table format: The first five column are fixed as chromosome name (CHROM), position (POS), reference nucleotide (REF), alter nucleotide (ALT) and additional information (INFO). Accession genotype should be in followed columns. “-” will be treated as Indel. “.” and “N” will be treated as missing data. Field in additional information column should be in format “tag=value”, and separated by semicolon “;”. Heterozygote should be looks like “A/G” or “A|G”.

Table 2: Table format of the genotypic dataset

Table 3 is an example of annotation file in BED6 format. As described at UCSC, the BED6 file contains 6 columns: 1) chromosome name, 2) chromosome start, 3) chromosome end, 4) name, 5) score and 6) strand. The BED4 contains the first 4 column of BED6.

BE NOTE THAT: the fourth column was used to define the name and types, which were separated by a space. For example, the first line of Table 3 indicates that: the genomic interval from 9154280 (exclude) to 9154821 (include) on Chr7 chromosome is CDS of “LOC_Os07g15770.1” and the strand is “negative”.

Table 3: An annotation example in BED6 format

Note: the red dot in fourth column indicate a space.

Table 4 is an example of detailed information of individuals, includes group/category, geo-coordinates and phenotype data. First column are names of accessions/individuals, phenotypic information are listed in followed columns.

Table 4: An example of accession detailed information dataset

1.2 Output data

The main results are hapResult and hapSummary class in R, consist of a matrix which could be divided into three parts as shown in Fig.1, and some additional attributes.

Cartoon representation of hapResult and hapSummary contents

Part I consists of only one column. And the first four lines were fixed as CHROM (chromosome name), POS (position), INFO (additional information) and ALLELE (allele). And followed lines are names of each haplotype. Part II consists of at least one column, contains site information (first four lines) and genotypes (followed lines). The part III of hapResult consists of one column named as Accession, while hapSummary consists of two columns named as Accession and freq (frequency of each haplotype).

The differences between hapResult and hapSummary is that each line of hapResult indicate an accession/individual, and each line in hapSummary indicate a haplotype.

Import VCF

# import vcf file
vcf <- import_vcf("your_vcf_file_path.vcf")

# import gziped vcf file
vcf <- import_vcf("your_vcf_file_path.vcf.gz")

Import p.link (ped/map)

plink <- import_plink.pedmap(mapfile = "p_link.map", pedfile = "p_link.ped", 
                             sep_ped = "\t", sep_map = "\t")
plink <- import_plink.pedmap(root = "p_link", sep_ped = "\t", sep_map = "\t")

Import GFF

# import GFFs
gff <- import_gff("your_gff_file_path.gff", format = "GFF")

Import BED

# import GFFs
bed <- import_bed("your_gff_file_path.bed")

Import DNA sequences

# import DNA sequences in fasta format
seqs <- import_seqs("your_DNA_seq_file_path.fa", format = "fasta")

VCF filtration

There are three modes to filter a vcfR object after import VCF into ‘R’: a. by position; b. by annotation; c. by both of them.

# filter VCF by position
vcf_f1 <- filter_vcf(vcf, mode = "POS",
                     Chr = "scaffold_1",
                     start = 4300, end = 5890)

# filter VCF by annotation
vcf_f2 <- filter_vcf(vcf, mode = "type",
                     gff = gff,
                     type = "CDS")

# filter VCF by position and annotation
vcf_f3 <- filter_vcf(vcf, mode = "both",
                     Chr = "scaffold_1",
                     start = 4300, end = 5890,
                     gff = gff,
                     type = "CDS")

It’s a time consuming work to import and manipulate a very large file with ‘R’ on personal computer. It’ll be more efficiency to extract the target ranges from origin VCF with filterLargeVCF() before import. If your VCF file is just a few ‘MB’, this step was not necessary at all.

Note: if extract more than one ranges, length of output file names (VCFout) must be equal with Chr and POS.

# new VCF file will be saved to disk
# extract a single gene/range from a large vcf

filterLargeVCF(VCFin = "Ori.vcf.gz",
               VCFout = "filtered.vcf.gz",
               Chr = "scaffold_8",
               POS = c(19802,24501),
               override = TRUE)

# extract multi genes/ranges from large vcf
filterLargeVCF(VCFin = "Ori.vcf.gz",          # surfix should be .vcf.gz or .vcf
               VCFout = c("filtered1.vcf.gz", # surfix should be .vcf.gz or .vcf
                          "filtered2.vcf.gz", 
                          "filtered3.vcf.gz"),
               Chr = c("scaffold_8",
                       "scaffold_8",
                       "scaffold_7"),
               POS = list(c(19802,24501), 
                          c(27341,28949),
                          c(38469,40344)),
               override = TRUE)               # if TRUE, existed file will be override without warning

p.link (ped/map) filtration

p.link <- filter_plink.pedmap(p.link, mode = "POS", 
                              Chr = "Chr08", start = 25947258, end = 25948258)

DNA sequences manipulation

The origin DNA sequences must be aligned and trimmed due to haplotype calculation need all sequences have same length. Those operations could be done with geneHapR. I still suggest users align and trim DNA sequences with Mega software and then save the result as FASTA format before import them into ‘R’.

# sequences alignment
seqs <- allignSeqs(seqs, quiet = TRUE)

# sequences trim
seqs <- trimSeqs(seqs, minFlankFraction = 0.1)
seqs

hapResult/hapSummary filtration

hap <- filter_hap(hapSummary,
                  rm.mode = c("position", "accession", "haplotype", "freq"),
                  position.rm = c(4879, 4950),
                  accession.rm = c("C1", "C9"),
                  haplotype.rm = c("H009", "H008"),
                  freq.min = 5)

Add annotations to hapResult

While hapResult was calculated from vcfR object, the INFO was taken from @fix field. The VCF INFO may missing some annotations. or contents format was inappropriate to display. Further more, INFO contents nothing if hapResult was generated from sequences. Here, we can introduce/replace the origin INFO by addINFO().

Note that: length of values must be equal with number of sites.

Let’s see how mant sites contains in the hapResult.

# Chech number of sites conclude in hapResult
sites(hapResult)

Now we replace the old INFO field with new tag named as “PrChange”.

# add annotations to INFO field
hapResult <- addINFO(hapResult,
                     tag = "PrChange",
                     values = rep(c("C->D", "V->R", "G->N"),3),
                     replace = TRUE)

Here, we add a tag named as “CDSChange” followed the old INFO.

# To replace the origin INFO by set 'replace' as TRUE
hapResult <- addINFO(hapResult,
                     tag = "CDSChange",
                     values = rep(c("C->A", "T->C", "G->T"),3),
                     replace = FALSE)

Adjust position of “ATG”

This function was only used to adjust the position of “ATG” to 0 and hence convert the gene on negative strand to positive strand.

Be note that: GFF and hapResult need to adjust position of ATG with the same parameters.

# set ATG position as zero in gff
newgff <- gffSetATGas0(gff = gff, hap = hapResult,
                       geneID = "test1G0387",
                       Chr = "scaffold_1",
                       POS = c(4300, 7910)) 

# set position of ATG as zero in hapResult/hapSummary
newhap <- hapSetATGas0(gff = gff, hap = hapResult,
                       geneID = "test1G0387",
                       Chr = "scaffold_1",
                       POS = c(4300, 7910))

Summary hapResult

Now, we have the hapResult object with INFOs we want display in next step. The hap_summary() function convert the object of hapResult class, which is a long table format, into a short table belong to hapSummary class. In hapResult each row represent a accession, while each row represents a hap in hapSummary.

hapSummary <- hap_summary(hapResult)
hapSummary

Visualize haplotye as table

Let’s see how to visualization of our haplotype results.

At first let’s display the hapSummary as a table. In this table like figure we can see all the variants and their positions, haplotypes and their frequencies.

plotHapTable(hapSummary)

Also we can add an annotation, “CDSChange”, to the table by assign the INFO_tag. It’s your responsibility to verify whether the INFO_tag was existed in the INFO field.

# add one annotation
plotHapTable(hapSummary,
             hapPrefix = "H",
             INFO_tag = "CDSChange", 
             tag_name = "CDS",
             displayIndelSize = 1, 
             angle = 45,
             replaceMultiAllele = TRUE,
             ALLELE.color = "grey90")

Now let’s add another INFO_tag named as “PrChange”.

# add multi annotation
plotHapTable(hapSummary,
             hapPrefix = "H",
             INFO_tag = c("CDSChange", "PrChange"),
             displayIndelSize = 1, 
             angle = 45,
             replaceMultiAllele = TRUE,
             ALLELE.color = "grey90")

Parameter tag_name was used to replace the character if INFO_tag was too long.

# add multi annotation
plotHapTable(hapSummary,
             hapPrefix = "H",
             INFO_tag = c("CDSChange", "PrChange"),
             tag_name = c("CDS", "Pr"),
             displayIndelSize = 1, 
             angle = 45,
             replaceMultiAllele = TRUE,
             ALLELE.color = "grey90")

Display variations on gene model.

I think it’s a good idea to figure out where are the variants by marking them on gene model.

displayVarOnGeneModel(hapSummary, gff,
                      Chr = "scaffold_1",
                      startPOS = 4300, endPOS = 7910,
                      type = "pin", cex = 0.7,
                      CDS_h = 0.05, fiveUTR_h = 0.02, threeUTR_h = 0.01)

hapNet calculation and visualization

The hapNet could be generated from object of hapSummary class. The accession group information could be attached in this step.

hapNet <- get_hapNet(hapSummary,
                     AccINFO = AccINFO,
                     groupName = "Type")

Once we have the hapNet object, we can plot it with ‘R’.

# plot haploNet

plotHapNet(hapNet,
           size = "freq",                   # circle size
           scale = "log2",                 # scale circle with 'log10(size + 1)'
           cex = 0.8,                       # size of hap symbol
           col.link = 2,                    # link colors
           link.width = 2,                  # link widths
           show.mutation = 2,               # mutation types one of c(0,1,2,3)
           legend = c(-12.5, 7))        # legend position

Phenotype association analysis

Finally, let’s see which haplotype has superiority at particular area by interact with phynotype.

Here are two options, merged or separated, to organized the heatmap of p-values and violin plot. The figure as an object of ggplot2, which means user could add/modified figure elements with ggplot2.

Here is an example for merged arrangement:

results <-hapVsPheno(hapResult,
                     hapPrefix = "H",
                     title = "This is title",
                     mergeFigs = TRUE,
                     pheno = pheno,
                     phenoName = "GrainWeight.2021",
                     minAcc = 3)
plot(results$figs)

An example for separated plot:

results <- hapVsPheno(hap = hapResult,
                      hapPrefix = "H",
                      title = "This is title",
                      pheno = pheno,
                      phenoName = "GrainWeight.2021",
                      minAcc = 3,
                      mergeFigs = FALSE)
plot(results$fig_pvalue)
plot(results$fig_Violin)

Association analysis of multi-phenotypes once a time

I believe the function of hapVsPhenos() will be useful there are a lot of phenotype need to be associated with haplotype results.

Note that: the pheno name will be added between the file name and surfix.

hapVsPhenos(hapResult,
            pheno,
            outPutSingleFile = TRUE,
            hapPrefix = "H",
            title = "Seita.0G000000",
            file = "mypheno.tiff",
            width = 12,
            height = 8,
            res = 300)