--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/VCF2Hapmap/VCF2FastaAndHapmap.pl	Wed Feb 07 22:08:47 2018 -0500
@@ -0,0 +1,525 @@
+
+#!/usr/bin/perl
+
+use strict;
+use Getopt::Long;
+
+my $usage = qq~Usage:$0 <args> [<opts>]
+
+where <args> are:
+
+    -v, --vcf          <VCF input>
+    -o, --out          <Output basename>
+      
+<opts> are:
+
+    -r, --reference    <Reference fasta file>
+    -g, --gff          <GFF input file to create alignments of genes>
+~;
+$usage .= "\n";
+
+my ($input,$out,$reference,$gff);
+
+
+
+GetOptions(
+	"vcf=s"          => \$input,
+	"out=s"          => \$out,
+	"reference=s"    => \$reference,
+	"gff=s"          => \$gff
+);
+
+
+die $usage
+  if ( !$input || !$out);
+ 
+if ($gff && !$reference)
+{
+	die "You must provide a Fasta reference file when providing GFF annotation\n";
+}
+
+ 
+my %ref_sequences;  
+if ($reference)
+{
+	my $id;
+	my $sequence = "";
+	open(my $R,$reference) or die "cannot open file: $reference";
+	while(<$R>)
+	{
+		my $line =$_;
+		$line =~s/\n//g;
+		$line =~s/\r//g;
+		if ($line =~ />([^\s]+)/){
+			$ref_sequences{$id} = $sequence;
+			$id=$1;$sequence="";
+		}
+		else
+		{
+			$sequence .= $line;
+		}
+	}
+	close($R);
+	$ref_sequences{$id} = $sequence;
+}
+
+
+my %chr_of_gene;
+my %ann;
+if ($gff)
+{
+	open(my $G,$gff) or die "cannot open file: $gff";
+	while(<$G>)
+	{
+		my $line =$_;
+		$line =~s/\n//g;
+		$line =~s/\r//g;
+		my @i = split(/\t/,$line);
+		my $chr = $i[0];
+		my $feature = $i[2];
+		my $strand = $i[6];
+		my $start = $i[3];
+		my $stop = $i[4];
+		my $inf = $i[8];
+		if ($feature eq 'gene')
+		{
+			 if ($inf =~/Name=([\w\-\.]+)[;\s]*/){$inf = $1;}
+			$ann{$inf}{"start"}=$start;
+			$ann{$inf}{"stop"}=$stop;
+			$ann{$inf}{"strand"}=$strand;
+			$chr_of_gene{$inf} = $chr;
+		}
+	}
+	close($G);
+}
+
+
+
+my %IUPAC =
+(
+        '[A/G]'=> "R",
+        '[G/A]'=> "R",
+        '[C/T]'=> "Y",
+        '[T/C]'=> "Y",
+        '[T/G]'=> "K",
+        '[G/T]'=> "K",
+        '[C/G]'=> "S",
+        '[G/C]'=> "S",
+        '[A/T]'=> "W",
+        '[T/A]'=> "W",
+        '[A/C]'=> "M",
+        '[C/A]'=> "M",
+        '[C/A/T]'=> "H",
+        '[A/T/C]'=> "H",
+        '[A/C/T]'=> "H",
+        '[C/T/A]'=> "H",
+        '[T/C/A]'=> "H",
+        '[T/A/C]'=> "H",
+        '[C/A/G]'=> "V",
+        '[A/G/C]'=> "V",
+        '[A/C/G]'=> "V",
+        '[C/G/A]'=> "V",
+        '[G/C/A]'=> "V",
+        '[G/A/C]'=> "V",
+        '[C/T/G]'=> "B",
+        '[T/G/C]'=> "B",
+        '[T/C/G]'=> "B",
+        '[C/G/T]'=> "B",
+        '[G/C/T]'=> "B",
+        '[G/T/C]'=> "B",
+        '[T/A/G]'=> "D",
+        '[A/G/T]'=> "D",
+        '[A/T/G]'=> "D",
+        '[T/G/A]'=> "D",
+        '[G/T/A]'=> "D",
+        '[G/A/T]'=> "D",
+);
+
+my %snps_of_gene;
+my %snps_of_gene2;
+my %indiv_order;
+my $indiv_list;
+my %genotyping_infos;
+my $num_line = 0;
+my $genename_rank_in_snpeff = 4;
+
+my $find_annotations = `grep -c 'EFF=' $input`;
+
+open(my $HAPMAP,">$out.hapmap");
+print $HAPMAP "rs#	alleles	chrom	pos	gene	feature	effect	codon_change	amino_acid_change	MAF	missing_data";
+open(my $VCF,$input);
+while(<$VCF>)
+{
+	my $line = $_;
+	chomp($line);
+	my @infos = split(/\t/,$line);
+	
+	if (/^##INFO=\<ID=EFF/ && /Amino_Acid_length \| Gene_Name \| Transcript_BioType \| Gene_Coding/)
+	{
+		$genename_rank_in_snpeff = 8;
+	}
+
+	if (scalar @infos > 9)
+	{
+		if (/#CHROM/)
+		{
+			for (my $j=9;$j<=$#infos;$j++)
+			{
+				my $individu = $infos[$j];
+				$indiv_list .= "	$individu";
+				$indiv_order{$j} = $individu;
+			}
+			print $HAPMAP "$indiv_list\n";
+		}
+		elsif (!/^#/)
+		{
+			$num_line++;
+
+			my $chromosome = $infos[0];
+			my $chromosome_position = $infos[1];
+			my $ref_allele = $infos[3];
+			my $alt_allele = $infos[4];
+                    	
+			if ($ref_allele =~/\w\w+/)
+			{
+				$ref_allele = "A";
+				$alt_allele = "T";
+			}
+			elsif ($alt_allele =~/\w\w+/)
+			{
+				$ref_allele = "T";
+				$alt_allele = "A";
+			}
+			
+			my $info = $infos[7];
+			my $is_in_exon = "#";
+			my $is_synonyme = "#";
+			my $gene;
+			if ($find_annotations > 1)
+			{
+				$gene = "intergenic";
+			}
+			else
+			{
+				$gene = $chromosome;
+			}
+			my $modif_codon = "#";
+			my $modif_aa = "#";
+			my $geneposition;
+			if ($info =~/EFF=(.*)/)
+			{
+					my @annotations = split(",",$1);
+					foreach my $annotation(@annotations)
+					{
+						my ($syn, $additional) = split(/\(/,$annotation);
+
+						if ($syn =~/STREAM/){next;}
+
+						$is_in_exon = "exon";
+						if ($syn =~/UTR/)
+						{
+							$is_in_exon = $syn;
+						}
+						else
+						{
+							$is_synonyme = $syn;
+						}
+						my @infos_additional = split(/\|/,$additional);
+						$gene = $infos_additional[$genename_rank_in_snpeff];
+						$modif_codon = $infos_additional[2];
+						$modif_aa = $infos_additional[3];
+						
+						if ($syn =~/INTERGENIC/)
+						{
+							$is_synonyme = "#";
+							$gene = "intergenic";
+							$is_in_exon = "#";
+						}
+						elsif ($syn =~/SYNONYM/)
+						{
+							$is_in_exon = "exon";
+						}
+						elsif ($syn =~/INTRON/ or $syn =~/SPLICE_SITE_DONOR/)
+						{
+							$is_in_exon = "intron";
+							$is_synonyme = "#";
+						}
+						
+						
+						if ($modif_aa =~/(\w)(\d+)$/)
+						{
+							$modif_aa = "$1/$1";
+						}
+						elsif ($modif_aa =~/(\w)(\d+)(\w)/)
+						{
+							$modif_aa = "$1/$3";
+						}
+						if ($infos_additional[8] =~/Exon/)
+						{
+							$is_in_exon = "exon";
+						}
+						
+						if (!$modif_aa){$modif_aa="#";}
+						if (!$modif_codon){$modif_codon="#";}
+					}
+			}
+			$gene =~s/\.\d//g;
+			if ($ann{$gene}{"start"})
+			{
+				my $strand = $ann{$gene}{"strand"};
+				if ($strand eq '-')
+				{
+					$geneposition = $ann{$gene}{"stop"} - $chromosome_position;
+				}
+				else
+				{
+					$geneposition = $chromosome_position - $ann{$gene}{"start"};
+				}
+			}
+			#if ($info =~/GenePos=(\d+);/)
+			#{
+			#	$geneposition = $1;
+			#}
+			my $ratio_missing_data;
+			my $snp_frequency;
+			my $genotyping = "";
+			
+			if (2 > 1)
+			{
+				
+				$genotyping_infos{"ref"} = "$ref_allele$ref_allele";
+				
+				my %alleles_found;
+				my $nb_readable_ind = 0;
+				for (my $i = 9;$i <= $#infos;$i++)
+				{
+					my $dnasample = $indiv_order{$i};
+					my @infos_alleles = split(":",$infos[$i]);
+					my $genotype = $infos_alleles[0];
+					$genotype =~s/0/$ref_allele/g;
+
+					if ($alt_allele =~/,/)
+					{
+						my @alt_alleles = split(",",$alt_allele);
+						my $num_all = 1;
+						foreach my $alt_al(@alt_alleles)
+						{
+							$genotype =~s/$num_all/$alt_al/g;
+							$num_all++;
+						}
+					}
+					else
+					{
+						$genotype =~s/1/$alt_allele/g;
+					}
+					if ($genotype eq '.'){$genotype = "./.";}
+					$genotype =~s/\./N/g;
+					if ($genotype !~/N\/N/)
+					{
+						$nb_readable_ind++;
+					}
+					my @alleles;
+					if ($genotype =~/\//)
+					{
+						@alleles = split(/\//,$genotype);
+					}
+					else
+					{
+						@alleles = split(/\|/,$genotype);
+					}
+					$genotyping .= join("",@alleles) . "	";
+					$genotyping_infos{$dnasample} = join("",@alleles);
+					
+					foreach my $al(@alleles)
+					{
+						if ($al ne 'N'){$alleles_found{$al}++;}
+					}	
+				}
+				chop($genotyping);
+				
+				$snp_frequency = 0;
+					
+				my $max = 0;
+				my $min = 10000000;
+				my $total = 0;
+				foreach my $al(keys(%alleles_found))
+				{
+					my $nb = $alleles_found{$al};
+					$total+= $nb;
+					if ($nb > $max)
+					{
+						$max = $nb;
+					}
+					if ($nb < $min)
+					{
+						$min = $nb;
+					}
+				}
+				if ($total > 0)
+				{
+					$snp_frequency = sprintf("%.1f",($min/$total)*100);
+				}
+				
+				$ratio_missing_data = 100 - ($nb_readable_ind / ($#infos - 8)) * 100;
+				$ratio_missing_data = sprintf("%.1f",$ratio_missing_data);
+
+				foreach my $dna(keys(%genotyping_infos))
+				{
+					$snps_of_gene{$gene}{$geneposition}{$dna} = $genotyping_infos{$dna};
+				}
+			}
+			my $snp_type = "[$ref_allele/$alt_allele]";
+			$snps_of_gene2{$chromosome}{$chromosome_position} = $snp_type;
+			#print $HAPMAP "$chromosome:$chromosome_position\t$snp_type\t$chromosome\t$chromosome_position\t$gene:$geneposition\t$is_in_exon\t$is_synonyme\t$modif_codon\t$modif_aa\t$snp_frequency%\t$nb_readable_ind\t$genotyping\n";
+			print $HAPMAP "$chromosome:$chromosome_position\t$ref_allele/$alt_allele\t$chromosome\t$chromosome_position\t$gene:$geneposition\t$is_in_exon\t$is_synonyme\t$modif_codon\t$modif_aa\t$snp_frequency%\t$ratio_missing_data%\t$genotyping\n";
+		}
+	}
+}
+close($VCF);
+close($HAPMAP);
+
+
+if (!$reference){exit;}
+
+#################################################################
+# generate flanking sequences for Illumina VeraCode technology
+#################################################################
+open(my $FLANKING,">$out.flanking.txt");
+foreach my $seq(keys(%ref_sequences))
+{
+                        if ($snps_of_gene2{$seq})
+                        {
+                                my $refhash = $snps_of_gene2{$seq};
+                                my %hashreal = %$refhash;
+
+                                # create consensus
+                                my $refseq = $ref_sequences{$seq};
+                                my $consensus = "";
+                                my $previous = 0;
+                                foreach my $pos(sort {$a<=>$b} keys(%hashreal))
+                                {
+                                        my $length = $pos - $previous - 1;
+                                        $consensus .= substr($refseq,$previous,$length);
+                                        my $iupac_code = $IUPAC{$snps_of_gene2{$seq}{$pos}};
+                                        $consensus .= $iupac_code;
+                                        $previous = $pos;
+                                }
+                                my $length = length($refseq) - $previous;
+                                $consensus .= substr($refseq,$previous,$length);
+
+                                foreach my $pos(sort {$a<=>$b} keys(%hashreal))
+                                {
+                                        my $snp_name = "$seq-$pos";
+                                        my $flanking_length = 60;
+                                        my $length = $flanking_length;
+                                        my $start = $pos - $flanking_length - 1;
+                                        if ($pos <= $flanking_length)
+                                        {
+                                                $length = $pos - 1;
+                                                $start = 0;
+                                        }
+                                        my $sequence = substr($consensus,$start,$length);
+				
+                                        $sequence .= $snps_of_gene2{$seq}{$pos};
+                                        $sequence .= substr($consensus,$pos,$flanking_length);
+                                        print $FLANKING "$snp_name,$sequence,0,0,0,Project_name,0,diploid,Other,Forward\n";
+                                }
+                        }
+	}
+
+	close($FLANKING);
+
+	if (!$gff){exit;}
+
+	my @individuals_list = split(/\t/,$indiv_list);
+	if ((scalar @individuals_list * scalar keys(%snps_of_gene)) > 800000)
+	{
+		print "Sorry, too many sequences to manage ...\n";
+		exit;
+	}
+
+	open(my $ALIGN_EGGLIB,">$out.gene_alignment.fas");
+	my %alignments_ind;
+	foreach my $seq(keys(%snps_of_gene))
+	{
+		if ($snps_of_gene{$seq})
+		{
+			my $refhash = $snps_of_gene{$seq};
+			my %hashreal = %$refhash;
+
+			# get flanking sequences
+			my %flanking5;
+			my $start = $ann{$seq}{"start"};
+			my $stop = $ann{$seq}{"stop"};
+			my $strand = $ann{$seq}{"strand"};
+			my $genelength = $stop - $start+1;
+			my $chr = $chr_of_gene{$seq};
+		my $refseq = substr($ref_sequences{$chr},$start-1,$genelength);
+		if ($strand eq '-')
+		{
+			$refseq =~ tr /atcgATCG/tagcTAGC/; $refseq = reverse($refseq);
+		}	
+		#print "$seq $chr $start $stop $refseq \n";
+		my $previous = 0;
+		foreach my $pos(sort {$a<=>$b} keys(%hashreal))
+		{
+			my $length = $pos - $previous - 1;
+			$flanking5{$pos} = substr($refseq,$previous,$length);
+			$previous = $pos;
+		}
+		my $length = length($refseq) - $previous;
+		my $flanking3 = substr($refseq,$previous,$length);
+		foreach my $ind(@individuals_list)
+		{
+			my $nb_missing_data_for_this_individual = 0;
+			if ($ind)
+			{
+                                                my $alignment_for_ind = "";
+                                                my $seq_without_underscore = $seq;
+                                                $seq_without_underscore =~s/_//g;
+                                                $alignment_for_ind .= ">$seq_without_underscore" . "_$ind" . "_1\n";
+                                                foreach my $pos(sort {$a<=>$b} keys(%hashreal))
+                                                {
+                                                        $alignment_for_ind .= $flanking5{$pos};
+                                                        my $geno = $snps_of_gene{$seq}{$pos}{$ind};
+                                                        $geno =~s/N/?/g;
+                                                        if ($geno =~/\?/){$nb_missing_data_for_this_individual++;}
+                                                        my @alleles = split("",$geno);
+                                                        $alignment_for_ind .= $alleles[0];
+                                                        if ($alleles[0] eq $alleles[1])
+                                                        {
+                                                                $alignments_ind{$ind} .= $alleles[1];
+                                                        }
+                                                        else
+                                                        {
+                                                                my $snp_type = "[" . $alleles[0] . "/" . $alleles[1] . "]";
+                                                                $alignments_ind{$ind} .= $IUPAC{$snp_type};
+                                                        }
+                                                }
+                                                $alignment_for_ind .= $flanking3;
+						$alignment_for_ind .= "\n";
+			
+			
+                                                $alignment_for_ind .= ">$seq_without_underscore" . "_$ind" . "_2\n";
+                                                foreach my $pos(sort {$a<=>$b} keys(%hashreal))
+                                                {
+                                                        $alignment_for_ind .= $flanking5{$pos};
+                                                        my $geno = $snps_of_gene{$seq}{$pos}{$ind};
+                                                        $geno =~s/N/?/g;
+                                                        my @alleles = split("",$geno);
+                                                        $alignment_for_ind .= $alleles[1];
+                                                }
+                                                $alignment_for_ind .= $flanking3;
+						$alignment_for_ind .= "\n";
+                                                if (keys(%hashreal) != $nb_missing_data_for_this_individual)
+                                                {
+                                                        print $ALIGN_EGGLIB $alignment_for_ind;
+                                                }
+			}
+		}
+	}
+}
+close($ALIGN_EGGLIB);
+
+
+