gwaslab 3.4.46__py3-none-any.whl → 3.4.47__py3-none-any.whl

gwaslab/viz_plot_regional2.py

@@ -0,0 +1,792 @@
+import pandas as pd
+import matplotlib.pyplot as plt
+import matplotlib.ticker as ticker
+import matplotlib.patches as patches
+import seaborn as sns
+import numpy as np
+import copy
+import scipy as sp
+from pyensembl import EnsemblRelease
+from allel import GenotypeArray
+from allel import read_vcf
+from allel import rogers_huff_r_between
+import matplotlib as mpl
+from scipy import stats
+from mpl_toolkits.axes_grid1.inset_locator import inset_axes
+from mpl_toolkits.axes_grid1.inset_locator import mark_inset
+from adjustText import adjust_text
+from gtfparse import read_gtf
+from gwaslab.g_Log import Log
+from gwaslab.bd_common_data import get_chr_to_number
+from gwaslab.bd_common_data import get_number_to_chr
+from gwaslab.bd_common_data import get_recombination_rate
+from gwaslab.bd_common_data import get_gtf
+from matplotlib.colors import ListedColormap
+from matplotlib.colors import LinearSegmentedColormap
+import matplotlib.colors
+from matplotlib.colors import Normalize
+from matplotlib.patches import Rectangle
+
+def _plot_regional(
+    sumstats,
+    fig,
+    ax1,
+    ax3,
+    region,
+    vcf_path,
+    marker_size,
+    fontsize,
+    build,
+    chrom_df,
+    xtick_chr_dict,
+    cut_line_color,
+    vcf_chr_dict = None,
+    gtf_path="default",
+    gtf_chr_dict = get_number_to_chr(),
+    gtf_gene_name=None,
+    rr_path="default",
+    rr_header_dict=None,
+    rr_chr_dict = get_number_to_chr(),
+    rr_lim = (0,100),
+    rr_ylabel = True,
+    rr_title=None,
+    region_ld_legend=True,
+    region_title=None,
+    mode="mqq",
+    region_step = 21,
+    region_ref=None,
+    region_ref_index_dic = None,
+    #region_ref_second=None,
+    region_grid = False,
+    region_grid_line = {"linewidth": 2,"linestyle":"--"},
+    region_lead_grid = True,
+    region_lead_grid_line = {"alpha":0.5,"linewidth" : 2,"linestyle":"--","color":"#FF0000"},
+    region_title_args = None,
+    region_hspace=0.02,
+    region_ld_threshold = [0.2,0.4,0.6,0.8],
+    region_ld_colors = ["#E4E4E4","#020080","#86CEF9","#24FF02","#FDA400","#FF0000","#FF0000"],
+    region_marker_shapes=None,
+    palette=None,
+    region_recombination = True,
+    region_protein_coding=True,
+    region_flank_factor = 0.05,
+    track_font_family="Arial",
+    taf=[4,0,0.95,1,1],
+    # track_n, track_n_offset,font_ratio,exon_ratio,text_offset
+    tabix=None,
+    chrom="CHR",
+    pos="POS",
+    verbose=True,
+    log=Log()
+):  
+
+    # x axix: use i to plot (there is a gap between i and pos) 
+
+
+    # if regional plot : pinpoint lead , add color bar ##################################################
+    if (region is not None) :
+        # pinpoint lead
+        lead_ids = []
+        
+        for index, region_ref_single in enumerate(region_ref):
+            ax1, lead_id_single = _pinpoint_lead(sumstats = sumstats,
+                                        ax1 = ax1, 
+                                        region_ref=region_ref_single,
+                                        lead_color = palette[(index+1)*100 + len(region_ld_threshold)+2], 
+                                        marker_size= marker_size,
+                                        region_marker_shapes=region_marker_shapes,
+                                        log=log,verbose=verbose)
+            if lead_id_single is not None:
+                lead_ids.append(lead_id_single)       
+        
+        # update region_ref to variant rsID or variantID / skip NAs
+        new_region_ref = []
+        for i in range(len(lead_ids)):
+            if lead_ids[i] is None:
+                continue
+            if region_ref[i] is None:
+                if "rsID" in sumstats.columns:
+                    new_name = sumstats.loc[lead_ids[i],"rsID"]
+                elif "SNPID" in sumstats.columns:
+                    new_name = sumstats.loc[lead_ids[i],"SNPID"]
+                else:
+                    new_name = "chr{}:{}".format(sumstats.loc[lead_ids[i],"CHR"] , sumstats.loc[lead_ids[i],"POS"])
+                new_region_ref.append(new_name)
+                region_ref_index_dic[new_name] = region_ref_index_dic[region_ref[i]]
+                continue
+            else:
+                new_region_ref.append(region_ref[i])
+        region_ref = new_region_ref
+        ##########################################################################################################
+
+        ##########################################################################################################
+
+        if (vcf_path is not None) and region_ld_legend:
+            ## plot cbar
+            ax1, cbar = _add_ld_legend(sumstats=sumstats, 
+                            ax1=ax1, 
+                            region_ref=region_ref,
+                            region_ld_threshold=region_ld_threshold, 
+                            region_ref_index_dic=region_ref_index_dic,
+                            palette=palette)
+        else:
+            cbar=None
+
+        if region_title is not None:
+                ax1 = _add_region_title(region_title, ax1=ax1,region_title_args=region_title_args )
+    
+    ## recombinnation rate ##################################################       
+    if (region is not None) and (region_recombination is True):
+        ax4 = _plot_recombination_rate(sumstats = sumstats,
+                                        pos =pos,
+                                        region= region, 
+                                        ax1 = ax1, 
+                                        rr_path =rr_path, 
+                                        rr_chr_dict = rr_chr_dict, 
+                                        rr_header_dict =rr_header_dict, 
+                                        build= build,
+                                        rr_lim=rr_lim,
+                                        rr_ylabel=rr_ylabel)
+     
+    ## regional plot : gene track ######################################################################
+                # calculate offset
+    if (region is not None):
+        most_left_snp      = sumstats["i"].idxmin()
+        
+        # distance between leftmost variant position to region left bound
+        gene_track_offset  = sumstats.loc[most_left_snp,pos] - region[1]
+        
+        # rebase i to region[1] : the i value when POS=0
+        gene_track_start_i = sumstats.loc[most_left_snp,"i"] - gene_track_offset - region[1]
+        
+        lead_snp_ys = []
+        lead_snp_is = []
+        lead_snp_is_colors = []
+        
+        for i,lead_id_single in enumerate(lead_ids):
+            if lead_id_single is not None:
+                lead_snp_ys.append(sumstats.loc[lead_id_single,"scaled_P"] )
+                lead_snp_is.append(sumstats.loc[lead_id_single,"i"])
+                lead_color = palette[(region_ref_index_dic[region_ref[i]]+1)*100 + len(region_ld_threshold) +1] # consistent color
+                lead_snp_is_colors.append(lead_color)
+
+    if gtf_path is not None:
+        # load gtf
+        ax3, texts_to_adjust_middle =_plot_gene_track(
+                        ax3=ax3,
+                        fig=fig,
+                        gtf_path=gtf_path,
+                        region=region,
+                        region_flank_factor=region_flank_factor,
+                        region_protein_coding=region_protein_coding,
+                        region_lead_grid=region_lead_grid,
+                        region_lead_grid_line=region_lead_grid_line,
+                        lead_snp_is=lead_snp_is,
+                        gene_track_start_i=gene_track_start_i,
+                        gtf_chr_dict=gtf_chr_dict,
+                        gtf_gene_name=gtf_gene_name, 
+                        track_font_family=track_font_family,
+                        taf=taf,
+                        build=build, 
+                        verbose=verbose, 
+                        log=log)
+
+    ## regional plot - set X tick
+    if region is not None:
+        region_ticks = list(map('{:.3f}'.format,np.linspace(region[1], region[2], num=region_step).astype("int")/1000000)) 
+        
+        # set x ticks for gene track
+        if "r" in mode:
+            if gtf_path is not None: 
+                ax3.set_xticks(np.linspace(gene_track_start_i+region[1], gene_track_start_i+region[2], num=region_step))
+                ax3.set_xticklabels(region_ticks,rotation=45,fontsize=fontsize,family="sans-serif")
+            
+            if region_grid==True:
+                for i in np.linspace(gene_track_start_i+region[1], gene_track_start_i+region[2], num=region_step):
+                    ax1.axvline(x=i, color=cut_line_color,zorder=1,**region_grid_line)
+                    ax3.axvline(x=i, color=cut_line_color,zorder=1,**region_grid_line)
+            
+            if region_lead_grid==True:
+                for lead_snp_i,  lead_snp_y, lead_snp_is_color in zip(lead_snp_is, lead_snp_ys , lead_snp_is_colors):
+                    region_lead_grid_line["color"] = lead_snp_is_color
+                    ax1.plot([lead_snp_i,lead_snp_i],[0,lead_snp_y], zorder=1,**region_lead_grid_line)
+                    ax3.axvline(x=lead_snp_i, zorder=2,**region_lead_grid_line)
+
+        else:
+            # set x ticks m plot
+            ax1.set_xticks(np.linspace(gene_track_start_i+region[1], gene_track_start_i+region[2], num=region_step))
+            ax1.set_xticklabels(region_ticks,rotation=45,fontsize=fontsize,family="sans-serif")
+    
+        ax1.set_xlim([gene_track_start_i+region[1], gene_track_start_i+region[2]])
+
+    # gene track (ax3) text adjustment
+    if (gtf_path is not None ) and ("r" in mode):
+        if len(texts_to_adjust_middle)>0:
+            adjust_text(texts_to_adjust_middle,
+                    autoalign=False, 
+                    only_move={'points':'x', 'text':'x', 'objects':'x'},
+                    ax=ax3,
+                    precision=0,
+                    force_text=(0.1,0),
+                    expand_text=(1, 1),
+                    expand_objects=(1,1),
+                    expand_points=(1,1),
+                    va="center",
+                    ha='center',
+                    avoid_points=False,
+                    lim =1000)     
+    
+    return ax1, ax3, ax4, cbar, lead_snp_is, lead_snp_is_colors
+
+# + ###########################################################################################################################################################################
+def _get_lead_id(sumstats=None, region_ref=None, log=None, verbose=True):
+    region_ref_to_check = copy.copy(region_ref)
+    try: 
+        if len(region_ref_to_check)>0 and type(region_ref_to_check) is not str:
+            region_ref_to_check = region_ref_to_check[0]
+    except:
+        pass
+    
+    lead_id=None
+    
+    if "rsID" in sumstats.columns:
+        lead_id = sumstats.index[sumstats["rsID"] == region_ref_to_check].to_list()
+
+    if lead_id is None and "SNPID" in sumstats.columns:
+        lead_id = sumstats.index[sumstats["SNPID"] == region_ref_to_check].to_list()
+
+    if type(lead_id) is list:
+        if len(lead_id)>0:
+            lead_id = int(lead_id[0])
+    
+    if region_ref_to_check is not None:
+        if type(lead_id) is list:
+            if len(lead_id)==0 :
+                log.warning("{} not found.. Skipping..".format(region_ref_to_check))
+                #lead_id = sumstats["scaled_P"].idxmax()
+                lead_id = None
+                return lead_id
+        else:
+            log.write(" -Reference variant ID: {} - {}".format(region_ref_to_check, lead_id), verbose=verbose)
+
+    if lead_id is None:
+        log.write(" -Extracting lead variant...", verbose=verbose)
+        lead_id = sumstats["scaled_P"].idxmax()
+
+    return lead_id
+
+def _pinpoint_lead(sumstats,ax1,region_ref, lead_color, marker_size, log, verbose,region_marker_shapes):
+    
+    if region_ref is None:
+        log.write(" -Extracting lead variant..." , verbose=verbose)
+        lead_id = sumstats["scaled_P"].idxmax()
+    else:
+        lead_id = _get_lead_id(sumstats, region_ref, log, verbose)
+    
+    if lead_id is not None:
+        ax1.scatter(sumstats.loc[lead_id,"i"],sumstats.loc[lead_id,"scaled_P"],
+                color=lead_color,
+                zorder=3,
+                marker= region_marker_shapes[sumstats.loc[lead_id,"SHAPE"]-1],
+                s=marker_size[1]+2,
+                edgecolor="black")
+
+    return ax1, lead_id
+# -############################################################################################################################################################################
+def _add_region_title(region_title, ax1,region_title_args):
+    ax1.text(0.015,0.97, region_title, transform=ax1.transAxes, va="top", ha="left", region_ref=None, **region_title_args )
+    return ax1
+
+def _add_ld_legend(sumstats, ax1, region_ld_threshold, region_ref,region_ref_index_dic,palette =None, position=1):
+    
+    width_pct = "11%"
+    height_pct = "{}%".format( 14 + 7 * len(region_ref))
+    axins1 = inset_axes(ax1,
+            width=width_pct,  # width = 50% of parent_bbox width
+            height=height_pct,  # height : 5%
+            loc='upper right',axes_kwargs={"frameon":True,"facecolor":"white","zorder":999999})
+    
+    ld_ticks = [0]+region_ld_threshold+[1]
+
+    for index, ld_threshold in enumerate(ld_ticks):
+        for group_index in range(len(region_ref)):
+            if index < len(ld_ticks)-1:            
+                x=ld_threshold
+                y=0.2*group_index
+                width=0.2
+                height=ld_ticks[index+1]-ld_ticks[index]
+                hex_color = palette[(region_ref_index_dic[region_ref[group_index]]+1)*100 + index+1] # consistent color
+                
+                a = Rectangle((x,y),width, height, fill = True, color = hex_color , linewidth = 2)
+                #patches.append(a)
+                axins1.add_patch(a)
+
+    # y snpid
+    yticks_position = 0.1 + 0.2 *np.arange(0,len(region_ref))
+    axins1.set_yticks(yticks_position, ["{}".format(x) for x in region_ref])
+    axins1.set_ylim(0,0.2*len(region_ref))    
+    
+    # x ld thresholds
+    axins1.set_xticks(ticks=ld_ticks) 
+    axins1.set_xticklabels([str(i) for i in ld_ticks]) 
+    axins1.set_xlim(0,1)    
+
+    axins1.set_aspect('equal', adjustable='box')
+    axins1.set_title('LD $r^{2}$ with variant',loc="center",y=-0.2)
+    cbar = axins1
+    return ax1, cbar
+
+# -############################################################################################################################################################################
+def  _plot_recombination_rate(sumstats,pos, region, ax1, rr_path, rr_chr_dict, rr_header_dict, build,rr_lim,rr_ylabel=True):
+    ax4 = ax1.twinx()
+    most_left_snp = sumstats["i"].idxmin()
+    
+    # the i value when pos=0
+    rc_track_offset = sumstats.loc[most_left_snp,"i"]-sumstats.loc[most_left_snp,pos]
+
+    if rr_path=="default":
+        if rr_chr_dict is not None:
+            rr_chr = rr_chr_dict[region[0]]
+        else:
+            rr_chr = str(region[0])
+        rc = get_recombination_rate(chrom=rr_chr,build=build)
+    else:
+        rc = pd.read_csv(rr_path,sep="\t")
+        if rr_header_dict is not None:
+            rc = rc.rename(columns=rr_header_dict)
+
+    rc = rc.loc[(rc["Position(bp)"]<region[2]) & (rc["Position(bp)"]>region[1]),:]
+    ax4.plot(rc_track_offset+rc["Position(bp)"],rc["Rate(cM/Mb)"],color="#5858FF",zorder=1)
+    
+    ax1.set_zorder(ax4.get_zorder()+1)
+    ax1.patch.set_visible(False)
+    
+    if rr_ylabel:
+        ax4.set_ylabel("Recombination rate(cM/Mb)")
+    if rr_lim!="max":
+        ax4.set_ylim(rr_lim[0],rr_lim[1])
+    else:
+        ax4.set_ylim(0, 1.05 * rc["Rate(cM/Mb)"].max())
+    ax4.spines["top"].set_visible(False)
+    ax4.spines["top"].set(zorder=1) 
+    return ax4
+
+# -############################################################################################################################################################################
+def _plot_gene_track(
+    ax3,
+    fig,
+    gtf_path,
+    region,
+    region_flank_factor,
+    region_protein_coding,
+    region_lead_grid,
+    region_lead_grid_line,
+    lead_snp_is,
+    gene_track_start_i,
+    gtf_chr_dict,gtf_gene_name, 
+    track_font_family,
+    taf,
+    build, 
+    verbose=True, 
+    log=Log()):
+    
+    # load gtf
+    log.write(" -Loading gtf files from:" + gtf_path, verbose=verbose)
+    uniq_gene_region,exons = process_gtf(   gtf_path = gtf_path ,
+                                            region = region, 
+                                            region_flank_factor = region_flank_factor,
+                                            build=build,
+                                            region_protein_coding=region_protein_coding,
+                                            gtf_chr_dict=gtf_chr_dict,
+                                            gtf_gene_name=gtf_gene_name)
+
+    n_uniq_stack = uniq_gene_region["stack"].nunique()
+    stack_num_to_plot = max(taf[0],n_uniq_stack)
+    ax3.set_ylim((-stack_num_to_plot*2-taf[1]*2,2+taf[1]*2))
+    ax3.set_yticks([])
+    pixels_per_point = 72/fig.dpi
+    pixels_per_track = np.abs(ax3.transData.transform([0,0])[1] - ax3.transData.transform([0,1])[1])                   
+    font_size_in_pixels= taf[2] * pixels_per_track
+    font_size_in_points =  font_size_in_pixels * pixels_per_point
+    linewidth_in_points=   pixels_per_track * pixels_per_point
+    log.write(" -plotting gene track..", verbose=verbose)
+    
+    sig_gene_name = "Undefined"
+    sig_gene_name2 = "Undefined"
+    texts_to_adjust_left = []
+    texts_to_adjust_middle = []
+    texts_to_adjust_right = []
+    for index,row in uniq_gene_region.iterrows():
+
+        gene_color="#020080"
+        #if row[6][0]=="+":
+        if row["strand"][0]=="+":
+            gene_anno = row["name"] + "->"
+        else:
+            gene_anno = "<-" + row["name"] 
+        
+
+        sig_gene_names=[]
+        sig_gene_lefts=[]
+        sig_gene_rights=[]
+        for lead_snp_i in lead_snp_is:
+            if region_lead_grid is True and lead_snp_i > gene_track_start_i+row["start"] and lead_snp_i < gene_track_start_i+row["end"] :
+                    gene_color=region_lead_grid_line["color"]
+                    sig_gene_names.append(row["name"])
+                    sig_gene_lefts.append(gene_track_start_i+row["start"])
+                    sig_gene_rights.append(gene_track_start_i+row["end"])
+
+            # plot gene line
+            ax3.plot((gene_track_start_i+row["start"],gene_track_start_i+row["end"]),
+                        (row["stack"]*2,row["stack"]*2),color=gene_color,linewidth=linewidth_in_points/10)
+        
+        
+        # plot gene name
+        if row["end"] >= region[2]:
+            #right side
+            texts_to_adjust_right.append(ax3.text(x=gene_track_start_i+region[2],
+                    y=row["stack"]*2+taf[4],s=gene_anno,ha="right",va="center",color="black",style='italic', size=font_size_in_points,family=track_font_family))
+            
+        elif row["start"] <= region[1] :
+            #left side
+            texts_to_adjust_left.append(ax3.text(x=gene_track_start_i+region[1],
+                    y=row["stack"]*2+taf[4],s=gene_anno,ha="left",va="center",color="black",style='italic', size=font_size_in_points,family=track_font_family))
+        else:
+            texts_to_adjust_middle.append(ax3.text(x=(gene_track_start_i+row["start"]+gene_track_start_i+row["end"])/2,
+                    y=row["stack"]*2+taf[4],s=gene_anno,ha="center",va="center",color="black",style='italic',size=font_size_in_points,family=track_font_family))
+    
+    # plot exons
+    for index,row in exons.iterrows():
+        exon_color="#020080" 
+        for sig_gene_name, sig_gene_left, sig_gene_right in zip(sig_gene_names,sig_gene_lefts,sig_gene_rights):
+            if not pd.isnull(row["name"]):
+                if (region_lead_grid is True) and row["name"]==sig_gene_name:
+                    exon_color = region_lead_grid_line["color"]  
+                else:
+                    exon_color="#020080" 
+            elif gene_track_start_i+row["starts"] > sig_gene_left and gene_track_start_i+row["end"] < sig_gene_right:
+                exon_color = region_lead_grid_line["color"]  
+            else:
+                exon_color="#020080"
+
+        ax3.plot((gene_track_start_i+row["start"],gene_track_start_i+row["end"]),
+                    (row["stack"]*2,row["stack"]*2),linewidth=linewidth_in_points*taf[3],color=exon_color,solid_capstyle="butt")
+
+    log.write(" -Finished plotting gene track..", verbose=verbose)
+
+    return ax3,texts_to_adjust_middle
+
+# -############################################################################################################################################################################
+# Helpers    
+# -############################################################################################################################################################################
+def process_vcf(sumstats, 
+                vcf_path, 
+                region,
+                region_ref, 
+                #region_ref_second, 
+                log, 
+                verbose, 
+                pos ,
+                nea,
+                ea, 
+                region_ld_threshold, 
+                vcf_chr_dict,
+                tabix):
+
+    log.write("Start to load reference genotype...", verbose=verbose)
+    log.write(" -reference vcf path : "+ vcf_path, verbose=verbose)
+
+    # load genotype data of the targeted region
+    ref_genotype = read_vcf(vcf_path,region=vcf_chr_dict[region[0]]+":"+str(region[1])+"-"+str(region[2]),tabix=tabix)
+    if ref_genotype is None:
+        log.warning("No data was retrieved. Skipping ...")
+        ref_genotype=dict()
+        ref_genotype["variants/POS"]=np.array([],dtype="int64")
+    log.write(" -Retrieving index...", verbose=verbose)
+    log.write(" -Ref variants in the region: {}".format(len(ref_genotype["variants/POS"])), verbose=verbose)
+    # match sumstats pos and ref pos: 
+    # get ref index for its first appearance of sumstats pos
+     #######################################################################################
+    def match_varaint(x):
+        # x: "POS,NEA,EA"
+        if np.any(ref_genotype["variants/POS"] == x.iloc[0]):
+            # position match
+            if len(np.where(ref_genotype["variants/POS"] == x.iloc[0] )[0])>1:  
+            # multiple position matches
+                for j in np.where(ref_genotype["variants/POS"] == x.iloc[0])[0]:
+                # for each possible match, compare ref and alt
+                    if x.iloc[1] == ref_genotype["variants/REF"][j]:
+                        if x.iloc[2] in ref_genotype["variants/ALT"][j]:
+                            return j
+                    elif x.iloc[1] in ref_genotype["variants/ALT"][j]:
+                        if x.iloc[2] == ref_genotype["variants/REF"][j]:
+                            return j    
+                return None
+            else: 
+                # single match
+                return np.where(ref_genotype["variants/POS"] == x.iloc[0] )[0][0] 
+        else:
+            # no position match
+            return None
+    log.write(" -Matching variants using POS, NEA, EA ...", verbose=verbose)
+    #############################################################################################
+    sumstats["REFINDEX"] = sumstats[[pos,nea,ea]].apply(lambda x: match_varaint(x),axis=1)
+    #############################################################################################
+
+    #for loop to add LD information
+    #############################################################################################
+    for ref_n, region_ref_single in enumerate(region_ref):
+
+        rsq = "RSQ_{}".format(ref_n)
+        ld_single = "LD_{}".format(ref_n)
+        lead = "LEAD_{}".format(ref_n)
+        sumstats[lead]= 0
+
+        # get lead variant id and pos
+        if region_ref_single is None:
+            # if not specified, use lead variant
+            lead_id = sumstats["scaled_P"].idxmax()
+        else:
+            # figure out lead variant
+            lead_id = _get_lead_id(sumstats, region_ref_single, log, verbose)
+        
+        if lead_id is None:
+            sumstats[rsq] = None
+            sumstats[rsq] = sumstats[rsq].astype("float")
+            sumstats[ld_single] = 0             
+            continue
+
+        lead_pos = sumstats.loc[lead_id,pos]
+
+        # if lead pos is available: 
+        if lead_pos in ref_genotype["variants/POS"]:
+            
+            # get ref index for lead snp
+            lead_snp_ref_index = match_varaint(sumstats.loc[lead_id,[pos,nea,ea]])
+            #lead_snp_ref_index = np.where(ref_genotype["variants/POS"] == lead_pos)[0][0]
+
+            # non-na other snp index
+            other_snps_ref_index = sumstats["REFINDEX"].dropna().astype("int").values
+            # get genotype 
+            lead_snp_genotype = GenotypeArray([ref_genotype["calldata/GT"][lead_snp_ref_index]]).to_n_alt()
+            try:
+                if len(set(lead_snp_genotype[0]))==1:
+                    log.warning("The variant is mono-allelic in reference VCF. LD can not be calculated.")
+            except:
+                pass
+            other_snp_genotype = GenotypeArray(ref_genotype["calldata/GT"][other_snps_ref_index]).to_n_alt()
+            
+            log.write(" -Calculating Rsq...", verbose=verbose)
+            
+            if len(other_snp_genotype)>1:
+                valid_r2= np.power(rogers_huff_r_between(lead_snp_genotype,other_snp_genotype)[0],2)
+            else:
+                valid_r2= np.power(rogers_huff_r_between(lead_snp_genotype,other_snp_genotype),2)
+            sumstats.loc[~sumstats["REFINDEX"].isna(),rsq] = valid_r2
+        else:
+            log.write(" -Lead SNP not found in reference...", verbose=verbose)
+            sumstats[rsq]=None
+        
+        sumstats[rsq] = sumstats[rsq].astype("float")
+        sumstats[ld_single] = 0
+        
+        for index,ld_threshold in enumerate(region_ld_threshold):
+            # No data,LD = 0
+            # 0, 0.2  LD = 1
+            # 1, 0.4  LD = 2
+            # 2, 0.6  LD = 3
+            # 3, 0.8  LD = 4
+            # 4, 1.0  LD = 5
+            # lead    LD = 6
+
+            if index==0:
+                to_change_color = sumstats[rsq]>-1
+                sumstats.loc[to_change_color,ld_single] = 1
+            to_change_color = sumstats[rsq]>ld_threshold
+            sumstats.loc[to_change_color,ld_single] = index+2
+
+        sumstats.loc[lead_id,ld_single] = len(region_ld_threshold)+2
+        
+        sumstats.loc[lead_id,lead] = 1
+
+    ####################################################################################################    
+    final_shape_col = "SHAPE"
+    final_ld_col = "LD"
+    final_rsq_col = "RSQ"
+
+    sumstats[final_ld_col]  = 0
+    sumstats[final_shape_col] = 1
+    sumstats[final_rsq_col] = 0.0
+    
+    for i in range(len(region_ref)):
+        ld_single = "LD_{}".format(i)
+        current_rsq = "RSQ_{}".format(i)
+        a_ngt_b = sumstats[final_rsq_col] < sumstats[current_rsq]
+        #set levels with interval=100
+        sumstats.loc[a_ngt_b, final_ld_col] = 100 * (i+1) + sumstats.loc[a_ngt_b, ld_single]
+        sumstats.loc[a_ngt_b, final_rsq_col] = sumstats.loc[a_ngt_b, current_rsq]
+        sumstats.loc[a_ngt_b, final_shape_col] = i + 1
+
+    ####################################################################################################
+    log.write("Finished loading reference genotype successfully!", verbose=verbose)
+    return sumstats
+
+# -############################################################################################################################################################################
+
+def process_gtf(gtf_path,
+                region,
+                region_flank_factor,
+                build,
+                region_protein_coding,
+                gtf_chr_dict,
+                gtf_gene_name):
+    #loading
+    
+    # chr to string datatype using gtf_chr_dict
+    to_query_chrom = gtf_chr_dict[region[0]]
+
+    # loading gtf data
+    if gtf_path =="default" or gtf_path =="ensembl":
+        
+        gtf = get_gtf(chrom=to_query_chrom, build=build, source="ensembl")
+    
+    elif gtf_path =="refseq":
+
+        gtf = get_gtf(chrom=to_query_chrom, build=build, source="refseq")
+    
+    else:
+        # if user-provided gtf
+        #gtf = pd.read_csv(gtf_path,sep="\t",header=None, comment="#", low_memory=False,dtype={0:"string"})
+        gtf = read_gtf(gtf_path)
+        gtf = gtf.loc[gtf["seqname"]==gtf_chr_dict[region[0]],:]
+
+    # filter in region
+    genes_1mb = gtf.loc[(gtf["seqname"]==to_query_chrom)&(gtf["start"]<region[2])&(gtf["end"]>region[1]),:].copy()
+    
+    # extract biotype
+    #genes_1mb.loc[:,"gene_biotype"] = genes_1mb[8].str.extract(r'gene_biotype "([\w\.\_-]+)"')
+    
+    # extract gene name
+    if gtf_gene_name is None:
+        if gtf_path=="refseq":
+            #genes_1mb.loc[:,"name"] = genes_1mb[8].str.extract(r'gene_id "([\w\.-]+)"').astype("string")
+            genes_1mb.loc[:,"name"] = genes_1mb["gene_id"]
+        elif gtf_path =="default" or gtf_path =="ensembl":
+            #genes_1mb.loc[:,"name"] = genes_1mb[8].str.extract(r'gene_name "([\w\.-]+)"').astype("string")
+            genes_1mb.loc[:,"name"] = genes_1mb["gene_name"]
+        else:
+            #genes_1mb.loc[:,"name"] = genes_1mb[8].str.extract(r'gene_id "([\w\.-]+)"').astype("string")
+            genes_1mb.loc[:,"name"] = genes_1mb["gene_id"]
+    else:
+        #pattern = r'{} "([\w\.-]+)"'.format(gtf_gene_name)
+        #genes_1mb.loc[:,"name"] = genes_1mb[8].str.extract(pattern).astype("string")
+        genes_1mb.loc[:,"name"] = genes_1mb[gtf_gene_name]
+
+    # extract gene
+    #genes_1mb.loc[:,"gene"] = genes_1mb[8].str.extract(r'gene_id "([\w\.-]+)"')
+    genes_1mb.loc[:,"gene"] = genes_1mb["gene_id"]
+    
+
+    # extract protein coding gene
+    if region_protein_coding is True:
+        #genes_1mb  =  genes_1mb.loc[genes_1mb["gene_biotype"]=="protein_coding",:].copy()
+        pc_genes_1mb_list = genes_1mb.loc[(genes_1mb["feature"]=="gene")& (genes_1mb["gene_biotype"]=="protein_coding") & (genes_1mb["name"]!=""),"name"].values
+        genes_1mb = genes_1mb.loc[(genes_1mb["feature"].isin(["exon","gene"])) & (genes_1mb["name"].isin(pc_genes_1mb_list)),:]
+    # extract exon
+    exons = genes_1mb.loc[genes_1mb["feature"]=="exon",:].copy()
+    
+    #uniq genes
+    ## get all record with 2nd column == gene
+    #uniq_gene_region = genes_1mb.loc[genes_1mb[2]=="gene",:].copy()
+    uniq_gene_region = genes_1mb.loc[genes_1mb["feature"]=="gene",:].copy()
+    
+    ## extract region + flank
+    flank = region_flank_factor * (region[2] - region[1])
+    
+    ## get left and right boundary
+    #uniq_gene_region["left"] = uniq_gene_region[3]-flank
+    #uniq_gene_region["right"] = uniq_gene_region[4]+flank
+    #
+    uniq_gene_region["left"] = uniq_gene_region["start"]-flank
+    uniq_gene_region["right"] = uniq_gene_region["end"]+flank
+
+    # arrange gene track
+    stack_dic = assign_stack(uniq_gene_region.sort_values(["start"]).loc[:,["name","left","right"]])  
+
+    # map gene to stack and add stack column : minus stack
+    uniq_gene_region["stack"] = -uniq_gene_region["name"].map(stack_dic)
+    exons.loc[:,"stack"] = -exons.loc[:,"name"].map(stack_dic)
+
+    # return uniq_gene_region (gene records with left and right boundary)
+    # return exon records with stack number
+    return uniq_gene_region, exons
+
+
+# -############################################################################################################################################################################
+def assign_stack(uniq_gene_region):
+
+    stacks=[] ## stack : gene track
+    stack_dic={} # mapping gene name to stack
+    
+    for index,row in uniq_gene_region.iterrows():
+        if len(stacks)==0:
+            # add first entry 
+            stacks.append([(row["left"],row["right"])])
+            stack_dic[row["name"]] = 0
+        else:
+            for i in range(len(stacks)):
+                for j in range(len(stacks[i])):
+                    # if overlap
+                    if (row["left"]>stacks[i][j][0] and row["left"]<stacks[i][j][1]) or (row["right"]>stacks[i][j][0] and row["right"]<stacks[i][j][1]):
+                        # if not last stack : break
+                        if i<len(stacks)-1:
+                            break
+                        # if last stack : add a new stack
+                        else:
+                            stacks.append([(row["left"],row["right"])])
+                            stack_dic[row["name"]] = i+1
+                            break
+                    # if no overlap       
+                    else:
+                        # not last in a stack
+                        if j<len(stacks[i])-1:
+                            #if in the middle
+                            if row["left"]>stacks[i][j][1] and row["right"]<stacks[i][j+1][0]:
+                                stacks[i].insert(j+1,(row["left"],row["right"]))
+                                stack_dic[row["name"]] = i
+                                break
+                        #last one in a stack
+                        elif row["left"]>stacks[i][j][1]:
+                            stacks[i].append((row["left"],row["right"]))
+                            stack_dic[row["name"]] = i
+                            break
+                if row["name"] in stack_dic.keys():
+                    break         
+    return stack_dic
+
+def closest_gene(x,data,chrom="CHR",pos="POS",maxiter=20000,step=50):
+    gene = data.gene_names_at_locus(contig=x[chrom], position=x[pos])
+    if len(gene)==0:
+        i=0
+        while i<maxiter:
+            distance = i*step
+            gene_u = data.gene_names_at_locus(contig=x[chrom], position=x[pos]-distance)
+            gene_d = data.gene_names_at_locus(contig=x[chrom], position=x[pos]+distance)
+            if len(gene_u)>0 and len(gene_d)>0:
+                for j in range(0,step,1):
+                    distance = (i-1)*step
+                    gene_u = data.gene_names_at_locus(contig=x[chrom], position=x[pos]-distance-j)
+                    gene_d = data.gene_names_at_locus(contig=x[chrom], position=x[pos]+distance+j)
+                    if len(gene_u)>0:
+                        return -distance,",".join(gene_u)
+                    else:
+                        return distance,",".join(gene_d)
+            elif len(gene_u)>0:
+                return -distance,",".join(gene_u)
+            elif len(gene_d)>0:
+                return +distance,",".join(gene_d)
+            else:
+                i+=1
+        return distance,"intergenic"
+    else:
+        return 0,",".join(gene)