Commit 439606a3 authored by kai.b's avatar kai.b
Browse files

improved modular structure and more verbose

parent c20b9594

launch_universc.sh

+393 −352

File changed.

Preview size limit exceeded, changes collapsed.

sub/ExtractBasicStats.pl

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#!/usr/bin/perl



#########################################

#                                       #

#     Written by Kai Battenberg         #

#     Plant Symbiosis Research Team     #

#                                       #

#########################################



use strict;

use warnings;

use List::Util qw( min max sum);



#####Options#####

my $barcodefile=$ARGV[0];

my $adjust_length=$ARGV[1];

my $indir = $ARGV[2];

##########







#####Check input#####

#collect files needed

my $feature_file = $indir."/outs/raw_feature_bc_matrix/features.tsv.gz";

my $raw_barcode_file = $indir."/outs/raw_feature_bc_matrix/barcodes.tsv.gz";

my $filtered_barcode_file = $indir."/outs/filtered_feature_bc_matrix/barcodes.tsv.gz";

my $matrix_file = $indir."/outs/filtered_feature_bc_matrix/matrix.mtx.gz";

my $summary_file = $indir."/outs/metrics_summary.csv";

my $bam_file = $indir."/outs/possorted_genome_bam.bam";

my $out_file = $indir."/outs/basic_stats.txt";



if (! -e $indir) {

    die "Error: Data directory needs to be specified.\n";

}

elsif (! -e $feature_file || ! -e $raw_barcode_file || ! -e $filtered_barcode_file || ! -e $matrix_file || ! -e $summary_file || ! -e $bam_file) {

    die "Error: Iether selected directory is not a cellranger output or some critical files are missing.\n";

}

##########







#####MAIN#####

#make a hash of original and adjusted barcodes

my %adjusted2original;

open (ORIGINAL, "<$barcodefile") or die "cannot open $barcodefile.\n";

while (my $line = <ORIGINAL>) {

    my $original = $line;

    $original =~ s/\r//sig;

    $original =~ s/\n//sig;

    

    my $adjusted = $original;

    if ($adjust_length >= 0) {

        my $length = $adjust_length;

        $adjusted = substr ($length, 0, $adjusted);

    }

    else {

        my $length = abs ($adjust_length);

        my $As = "A" x $length;

        $adjusted = $As.$adjusted;

    }

    

    $adjusted2original{$adjusted} = $original;

}

close (ORIGINAL);



#gene count

print " getting gene count.\n";



my $ref_gene_count = `zcat $feature_file | wc -l`;

chomp $ref_gene_count;



#cell count

print " getting cell count.\n";



my $cellcount_raw;

$cellcount_raw = `zcat $raw_barcode_file | wc -l`;

chomp $cellcount_raw;



my $cellcount_filtered;

$cellcount_filtered = `zcat $filtered_barcode_file | wc -l`;

chomp $cellcount_filtered;



my %percell;

my $barcodes;

$barcodes = `zcat $filtered_barcode_file | cut -f1 -d'-'`;

chomp $barcodes;

my @barcodes = split (/\n/, $barcodes);



foreach my $barcode (@barcodes) {

    my %barcode;

    $percell{$barcode} = \%barcode;

}



#raw read count

print " getting raw read count.\n";



my $readcount_raw = `tail -n 1 $summary_file`;

chomp $readcount_raw;



my @readcount_raw = split (//, $readcount_raw);

my $quotestat = 0;

foreach my $character (@readcount_raw) {

	if ($character =~ m/\"/ && $quotestat == 0) {

		$character = "";

		$quotestat = 1;

		next;

	}

	elsif ($character =~ m/\"/ && $quotestat == 1) {

		$character = "";

		$quotestat = 0;

		next;

	}

	elsif ($character =~ m/\,/ && $quotestat == 1) {

		$character = "";

		next;

	}

	else {

		next;

	}

}

$readcount_raw = join ("", @readcount_raw);

$readcount_raw = (split (/\,/, $readcount_raw))[3];



#assigned read count

print " getting assigned read count.\n";



my $assigned_reads_percell = `samtools view $bam_file | grep 'CB:Z:' | sed 's\/CB:Z:\\([ACGT]*\\).*/\\1/' | awk '{print \$1, \$NF}' | sort | uniq | cut -f2 -d' ' | sort | uniq -c`;

chomp $assigned_reads_percell;

my @assigned_reads_percell = split (/\n/, $assigned_reads_percell);



foreach my $barcode (@assigned_reads_percell) {

    my $count = $barcode;

    $count =~ s/ +/ /g;

    $count =~ s/^ //;

    $count =~ s/ $//;

    my @count = split (/ /, $count);

    

    if (exists $percell{ $adjusted2original{$count[1]} }) {

        my %count;

        $count{ASSIGNED} = $count[0];

        $percell{ $adjusted2original{$count[1]} } = \%count;

    }

}

foreach my $barcode (sort keys %percell) {

    my %barcode = %{ $percell{$barcode} };

    if (!$barcode{ASSIGNED}) {

        $barcode{ASSIGNED} = 0;

    }

    $percell{$barcode} = \%barcode;

}



#mapped read count

print " getting mapped read count.\n";



my $mapped_reads_percell = `samtools view -F 4 $bam_file | grep 'CB:Z:' | sed 's\/CB:Z:\\([ACGT]*\\).*/\\1/' | awk '{print \$1, \$NF}' | sort | uniq | cut -f2 -d' ' | sort | uniq -c`;

chomp $mapped_reads_percell;

my @mapped_reads_percell = split (/\n/, $mapped_reads_percell);



foreach my $barcode (@mapped_reads_percell) {

    my $count = $barcode;

    $count =~ s/ +/ /g;

    $count =~ s/^ //;

    $count =~ s/ $//;

    my @count = split (/ /, $count);



    if (exists $percell{ $adjusted2original{$count[1]} }) {

        my %count = %{ $percell{ $adjusted2original{$count[1]} } };

        $count{MAPPED} = $count[0];

        $percell{ $adjusted2original{$count[1]} } = \%count;

    }

}

foreach my $barcode (sort keys %percell) {

    my %barcode = %{ $percell{$barcode} };

    if (!$barcode{MAPPED}) {

        $barcode{MAPPED} = 0;

    }

    $percell{$barcode} = \%barcode;

}



#UMI count

print " getting mapped UMI count.\n";



my %barcodes;

my $id = 0;

foreach my $barcode (@barcodes) {

    $id++;

    $barcodes{$id} = $barcode;

}



my $umicount = `zcat $matrix_file | tail -n +4 | cut -f2,3 -d ' '`;

chomp $umicount;

my @umicount = split (/\n/, $umicount);

my %umicount;

foreach my $entry (@umicount) {

    my @data = split (/ /, $entry);

    if (!exists $umicount{$data[0]}) {

        $umicount{$data[0]} = $data[1];

    }

    else {

        $umicount{$data[0]} = $umicount{$data[0]} + $data[1];

    }

}

foreach my $id (keys %umicount) {

    if (exists $percell{ $barcodes{$id} }) {

        my %data = %{ $percell{ $barcodes{$id} } };

        $data{UMI} = $umicount{$id};

        

        $percell{ $barcodes{$id} } = \%data;

    }

}

foreach my $barcode (sort keys %percell) {

    my %barcode = %{ $percell{$barcode} };

    if (!$barcode{UMI}) {

        $barcode{UMI} = 0;

    }

    $percell{$barcode} = \%barcode;

}



#Gene count per cell

print " getting gene count per cell.\n";



my @genecount = @umicount;

my %genecount;

foreach my $entry (@genecount) {

    my @data = split (/ /, $entry);

    if (!exists $genecount{$data[0]}) {

        $genecount{$data[0]} = 1;

    }

    else {

        $genecount{$data[0]}++;

    }

}

foreach my $id (keys %genecount) {

    if (exists $percell{ $barcodes{$id} }) {

        my %data = %{ $percell{ $barcodes{$id} } };

        $data{GENE} = $genecount{$id};

        

        $percell{ $barcodes{$id} } = \%data;

    }

}

foreach my $barcode (sort keys %percell) {

    my %barcode = %{ $percell{$barcode} };

    if (!$barcode{GENE}) {

        $barcode{GENE} = 0;

    }

    $percell{$barcode} = \%barcode;

}



#Total gene count

print " getting total gene count.\n";



my $genes_total = `zcat $matrix_file | tail -n +4 | cut -f1 -d ' ' | sort | uniq | wc -l`;

chomp $genes_total;



#Total, means, and medinans

print " getting total, mean, and medians.\n";



my @reads_assigned;

my @reads_mapped;

my @umis;

my @genes;

foreach my $barcode (keys %percell) {

    my %barcode = %{ $percell{$barcode} };

    push (@reads_assigned, $barcode{ASSIGNED});

    push (@reads_mapped, $barcode{MAPPED});

    push (@umis, $barcode{UMI});

    push (@genes, $barcode{GENE});

}



my $reads_assigned_total = sum (@reads_assigned);

my $reads_assigned_mean = &mean (@reads_assigned);

my $reads_assigned_median = &median (@reads_assigned);



my $reads_mapped_total = sum (@reads_mapped);

my $reads_mapped_mean = &mean (@reads_mapped);

my $reads_mapped_median = &median (@reads_mapped);



my $umis_total = sum (@umis);

my $umis_mean = &mean (@umis);

my $umis_median = &median (@umis);



my $genes_mean = &mean (@genes);

my $genes_median = &median (@genes);



#getting percentages

print " getting percentages.\n";



my $percent_assigned = sprintf("%.4f", $reads_assigned_total/$readcount_raw)*100;

$percent_assigned = $percent_assigned."\%";

my $percent_mapped = sprintf("%.4f", $reads_mapped_total/$reads_assigned_total)*100;

$percent_mapped = $percent_mapped."\%";

my $reads_per_umi = sprintf("%.2f", $reads_mapped_total/$umis_total);



#output

print " printing out summary.\n";



open (OUT, ">$out_file") or die "cannot open $out_file.\n";

print OUT "\#\#\#\#\#BASIC STATS\#\#\#\#\#\n";

print OUT "Reference gene count:\t$ref_gene_count\n";

print OUT "Cell count:\t(raw)$cellcount_raw\t(filtered)$cellcount_filtered\n";

print OUT "Read count(raw):\t$readcount_raw\n";

print OUT "Read count(assigned):\t(total)$reads_assigned_total\t(per cell mean)$reads_assigned_mean\t(per cell median)$reads_assigned_median\n";

print OUT "Read count(mapped):\t(total)$reads_mapped_total\t(per cell mean)$reads_mapped_mean\t(per cell median)$reads_mapped_median\n";

print OUT "UMI count(mapped to gene):\t(total)$umis_total\t(per cell mean)$umis_mean\t(per cell median)$umis_median\n";

print OUT "Gene count:\t(total)$genes_total\t(per cell mean)$genes_mean\t(per cell median)$genes_median\n";

print OUT "\%Reads assigned/raw:\t$percent_assigned\n";

print OUT "\%Reads mapped/assigned:\t$percent_mapped\n";

print OUT "reads/UMI:\t$reads_per_umi\n";

print OUT "\n";

print OUT "\#\#\#\#\#PER CELL\#\#\#\#\#\n";

print OUT "barcode\tRead count (assigned)\tRead count (mapped)\tUMI count(mapped)\tGene count\n";

foreach my $barcode (sort keys %percell) {

    my %barcode = %{ $percell{$barcode} };

    print OUT "$barcode\t$barcode{ASSIGNED}\t$barcode{MAPPED}\t$barcode{UMI}\t$barcode{GENE}\n";

}

close (OUT);

##########







#####SUB#####

sub mean {

    my $raw = sum(@_)/@_;

    my $round = sprintf("%.1f", $raw); 

    return $round;

}



sub median {

    my @vals = sort {$a <=> $b} @_;

    my $len = @vals;

    if($len%2) {

        return $vals[int($len/2)];

    }

    else {

        return ($vals[int($len/2)-1] + $vals[int($len/2)])/2;

    }

}

##########

__END__

sub/MakeIndropBarcodes.pl

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#!/usr/bin/perl



#####################################

#                                   #

#     Written by Kai Battenberg     #

#     Plant Sciences UC Davis       #

#                                   #

#####################################



use strict;

use warnings;



#####Options#####

my $gel_list1 = $ARGV[0];

my $gel_list2 = $ARGV[1];

my $version = $ARGV[2];

my $out = $ARGV[3];

##########







#####MAIN#####

my $list1 = `cat $gel_list1`;

chomp $list1;

my @list1 = split (/\n/, $list1);



my $list2 = `cat $gel_list2`;

chomp $list2;

my @list2 = split (/\n/, $list2);



my $length = scalar (@list1);







my @list12;

if ($version =~ m/v2/) {

    for (my $i = 0; $i < $length; $i++) {

        my $element1 = substr ($list1[$i], 0, 8);

        my $element12 = $element1.$list2[$i];

        push (@list12, $element12);

    }

}

elsif ($version =~ m/v3/) {

    foreach my $element1 (@list1) {

        $element1 = substr ($element1, 0, 8);

        foreach my $element2 (@list2) {

            my $element12 = $element1.$element2;

            push (@list12, $element12);

        }

    }

}



my %list12;

foreach my $element12 (sort @list12) {

    if (!exists $list12{$element12}) {

        $list12{$element12} = 1;

    }

    else {

        $list12{$element12}++

    }

}



my $outfile = $out."/inDrop-".$version."_barcodes.txt";

open (OUT, ">$outfile") or die "cannot open $outfile.\n";

foreach my $element12 (sort keys %list12) {

    if ($list12{$element12} < 2) {

        print OUT "$element12\n";

    }

}

close (OUT);

##########

__END__

sub/RecoverBarcodes.pl

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#!/usr/bin/perl



#########################################

#                                       #

#     Written by Kai Battenberg         #

#     Plant Symbiosis Research Team     #

#                                       #

#########################################



use strict;

use warnings;



#####Options#####

my $barcodefile = $ARGV[0];

my $adjust_length = $ARGV[1];

my $cellranger_dir = $ARGV[2];

##########







#####Main#####

#make a hash of original and adjusted barcodes

my %barcodes;

open (ORIGINAL, "<$barcodefile") or die "cannot open $barcodefile.\n";

while (my $line = <ORIGINAL>) {

    my $original = $line;

    $original =~ s/\r//sig;

    $original =~ s/\n//sig;

    

    my $adjusted = $original;

    if ($adjust_length >= 0) {

        my $length = $adjust_length;

        $adjusted = substr ($length, 0, $adjusted);

    }

    else {

        my $length = abs ($adjust_length);

        my $As = "A" x $length;

        $adjusted = $As.$adjusted;

    }

    

    $barcodes{$adjusted} = $original;

}

close (ORIGINAL);



#get modified barcode list (raw)

my $raw_file = $cellranger_dir."/outs/raw_feature_bc_matrix/barcodes.tsv.gz";

my $modified_barcodes_raw = `zcat $raw_file | cut -f1 -d'-'`;

$modified_barcodes_raw =~ s/\n$//;

my @modified_barcodes_raw = split (/\n/, $modified_barcodes_raw);



#get modified barcode list (filtered)

my $filtered_file = $cellranger_dir."/outs/filtered_feature_bc_matrix/barcodes.tsv.gz";

my $modified_barcodes_filtered = `zcat $filtered_file | cut -f1 -d'-'`;

$modified_barcodes_filtered =~ s/\n$//;

my @modified_barcodes_filtered = split (/\n/, $modified_barcodes_filtered);



#replace barcodes

my $temp = "barcodes.txt";

open (TEMP, ">$temp") or die "cannot open $temp.\n";

foreach my $barcode (@modified_barcodes_raw) {

    print TEMP "$barcodes{$barcode}-1\n";

}

close (TEMP);

system "gzip -c $temp > $raw_file";



open (TEMP, ">$temp") or die "cannot open $temp.\n";

foreach my $barcode (@modified_barcodes_filtered) {

    print TEMP "$barcodes{$barcode}-1\n";

}

close (TEMP);

system "gzip -c $temp > $filtered_file";

unlink ($temp);

##########

whitelists/AllPossibilities_2_barcodes.txt

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AA

AC

AG

AT

CA

CC

CG

CT

GA

GC

GG

GT

TA

TC

TG

TT

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