├── .gitignore ├── 1.calc_gene_exon_depth ├── DMD.NM_000109.png ├── DMD.NM_000109.txt ├── calc_gene_exon_depth.pl ├── draw_exon_coverage.R └── draw_exon_coverage.sh ├── 2.chrX-chrY ├── readme.md ├── simulate.pl └── snp-calling.sh ├── 3.draw_gene_structure ├── ANXA1-gene-structure.jpg ├── step1.gtf2mysql_format.pl ├── step2.create_gtf_table.sql ├── step3.upload_gtf2mysql.R └── step4.draw_gene_structure_from_mysql.R ├── LICENSE └── README.md /.gitignore: -------------------------------------------------------------------------------- 1 | /blib/ 2 | /.build/ 3 | _build/ 4 | cover_db/ 5 | inc/ 6 | Build 7 | !Build/ 8 | Build.bat 9 | .last_cover_stats 10 | /Makefile 11 | /Makefile.old 12 | /MANIFEST.bak 13 | /META.yml 14 | /META.json 15 | /MYMETA.* 16 | nytprof.out 17 | /pm_to_blib 18 | *.o 19 | *.bs 20 | /_eumm/ 21 | -------------------------------------------------------------------------------- /1.calc_gene_exon_depth/DMD.NM_000109.png: -------------------------------------------------------------------------------- https://raw.githubusercontent.com/jmzeng1314/my-perl/c865748b97d4565144cbd032669b45fa229d0855/1.calc_gene_exon_depth/DMD.NM_000109.png -------------------------------------------------------------------------------- /1.calc_gene_exon_depth/calc_gene_exon_depth.pl: -------------------------------------------------------------------------------- 1 | ############################################# 2 | #Author: Jianming Zeng 3 | #email: jmzeng1314@163.com 4 | #Creat Time: Mon Feb 1 10:13:18 EST 2016 5 | #URL1: http://www.bio-info-trainee.com/ 6 | #URL2: https://github.com/jmzeng1314 7 | #Eli Lilly and Company (CHINA LCRDC) 8 | ############################################# 9 | #use strict; 10 | #use warnings; 11 | use Getopt::Long; 12 | #View each exon covarage by giving a gene symbol 13 | 14 | #my $refgene='/home/jmzeng/ref-database/hg19_refGene.txt'; 15 | #my $bam='/home/jmzeng/aws_data/lichun/trash/L.bam'; 16 | #my $symbol='DMD'; 17 | my $help=undef; 18 | my $usage=<\$symbol, 30 | "r|refgene=s"=>\$refgene, 31 | "b|bam=s"=>\$bam, 32 | "h|help"=>\$help); 33 | 34 | #Start parameter checking 35 | 36 | # we can't put $ sign in the << charactor 37 | my $R_code=<draw_exon_coverage.R"; 67 | print FH $R_code; 68 | close FH; 69 | } 70 | =pod 71 | # check whether samtools in your $PATH 72 | unless(which('samtools')){ 73 | print <){ 98 | my @F=split; 99 | push @NM_list,$_ if $F[12] eq $symbol; 100 | } 101 | close FH; 102 | my $tmp_N=scalar(@NM_list); 103 | if ($tmp_N>0){ 104 | print "we have find $tmp_N mRNA(refseq database) for the gene you give !\n" 105 | } 106 | else{ 107 | print "we can't find any information for $symbol in $refgene \n"; 108 | exit(1); 109 | } 110 | undef $tmp_N; 111 | foreach (@NM_list){ 112 | my (undef,$NM_id,$chr,$strand,$start,$end,undef,undef, 113 | $exon_n,$up,$down,undef,$symbol,undef,undef,undef)=split; 114 | open FH,">$symbol.$NM_id.txt"; 115 | my @pos=split/,/,"$up,$down"; 116 | #exit; 117 | undef %h_depth; 118 | foreach ($start..$end){ 119 | $h_depth{$_}=0; 120 | } 121 | $command="samtools depth -r $chr:$start-$end $bam "; 122 | #print "$command\n"; 123 | my @depths=`$command`; 124 | foreach (@depths){ 125 | my @F=split; 126 | $h_depth{$F[1]}=$F[2]; 127 | } 128 | #print "$_\t$h_depth{$_}\t$group{$_}\n" foreach $start..$end; 129 | 130 | my @pos=sort @pos; 131 | #print join",",@pos; 132 | undef %group; 133 | foreach my $i (2..$#pos){ 134 | next if $i%2==1; 135 | foreach my $j ($pos[$i-1]..$pos[$i]){ 136 | my $n=$j-$pos[$i-1]+1; 137 | if ($strand eq '+'){ 138 | $exon=$i/2; 139 | }else{ 140 | $exon=$exon_n-$i/2+1; 141 | } 142 | #my $label="exon:$exon:$chr:$pos[$i-1]-$pos[$i]"; 143 | my $label="exon:$exon"; 144 | print FH "$n\t$h_depth{$j}\t$label\n"; 145 | } 146 | } 147 | close FH; 148 | my $cmd="Rscript draw_exon_coverage.R $symbol.$NM_id.txt"; 149 | #print $cmd."\n"; 150 | system($cmd) 151 | } 152 | #system("mkdir $symbol"); 153 | #system("mv $symbol.* $symbol"); 154 | print "[",scalar(localtime),"] Finished\n"; 155 | 156 | -------------------------------------------------------------------------------- /1.calc_gene_exon_depth/draw_exon_coverage.R: -------------------------------------------------------------------------------- 1 | args <- commandArgs(trailingOnly = TRUE) 2 | file=args[1] 3 | outpng=sub(".txt",".png",file) 4 | dat=read.table(file) 5 | names(dat)=c('pos','depth','exon') 6 | dat$exon=factor(dat$exon) 7 | library(ggplot2) 8 | png(outpng,width = 1080, height = 1080) 9 | p=ggplot(data=dat,aes(x=pos,y=depth,color=exon))+geom_line() 10 | p=p+facet_wrap(~exon,scales="free_x") 11 | p=p+theme(legend.position='none') 12 | print(p) 13 | dev.off() 14 | -------------------------------------------------------------------------------- /1.calc_gene_exon_depth/draw_exon_coverage.sh: -------------------------------------------------------------------------------- 1 | for i in *txt 2 | do 3 | echo $i 4 | Rscript draw_exon_coverage.R $i 5 | done 6 | -------------------------------------------------------------------------------- /2.chrX-chrY/readme.md: -------------------------------------------------------------------------------- 1 | # 从fasta序列里面模拟测序的reads走SNP-calling流程 2 | 3 | 很简单的一个shell脚本，从UCSC里面单独下载X,Y染色体的fasta序列，写脚本从Y染色体序列里面模拟双端测序的fastqa文件，然后用bwa软件比对到X染色体，作为参考基因组。全部代码如下： 4 | 5 | ```shell 6 | mkdir -p ~/tmp/chrX_Y/hg19/ 7 | cd ~/tmp/chrX_Y/hg19/ 8 | #conda install -c bioconda bwa 9 | #conda install -c bioconda samtools 10 | wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/chrX.fa.gz; 11 | wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/chrY.fa.gz; 12 | gunzip chrX.fa.gz 13 | gunzip chrY.fa.gz 14 | wget https://raw.githubusercontent.com/jmzeng1314/my-perl/master/2.chrX-chrY/simulate.pl 15 | bwa index chrX.fa 16 | perl simulate.pl chrY.fa 17 | bwa mem -t 5 -M chrX.fa read*.fa >read.sam 18 | samtools view -bS read.sam >read.bam 19 | samtools flagstat read.bam 20 | samtools sort -@ 5 -o read.sorted.bam read.bam 21 | samtools view -h -F4 -q 5 read.sorted.bam |samtools view -bS |samtools rmdup - read.filter.rmdup.bam 22 | samtools index read.filter.rmdup.bam 23 | samtools mpileup -ugf ~/tmp/chrX_Y/hg19/chrX.fa read.filter.rmdup.bam |bcftools call -vmO z -o read.bcftools.vcf.gz 24 | ``` 25 | 26 | 如果samtools安装的是最新版，上面的代码还可以更简化。 27 | 28 | 首先下载X,Y染色体的fasta序列，在UCSC上面下载即可。 29 | 然后把X染色体构建bwa的索引 30 | 接着模拟一个Y染色体的测序数据，模拟的程序很简单,模拟Y染色体的测序片段（PE100，insert400） 31 | 最后把模拟测序数据比对到X染色体的参考，统计一下比对结果即可！ 32 | **我自己看sam文件也发现真的同源性好高呀，总共就模拟了380万reads，就有120万是百分百比对上了。** 33 | 所以对女性个体来说，测序判断比对到Y染色体是再正常不过的了。如果要判断性别，必须要找那些X,Y差异性区段！对男性来说，更是如此！ 34 | 35 | 其中里面有一个perl代码，从Y染色体序列里面模拟双端测序的fastqa文件，需要仔细理解。 36 | 37 | ```perl 38 | while(<>){ 39 | chomp; 40 | $chrY.=uc $_; 41 | } 42 | $j=0; 43 | open FH_L,">read1.fa"; 44 | open FH_R,">read2.fa"; 45 | foreach (1..4){ 46 | for ($i=600;$i<(length($chrY)-600);$i = $i+50+int(rand(10))){ 47 | $up = substr($chrY,$i,100); 48 | $down=substr($chrY,$i+400,100); 49 | next unless $up=~/[ATCG]/; 50 | next unless $down=~/[ATCG]/; 51 | $down=reverse $down; 52 | $down=~tr/ATCG/TAGC/; 53 | $j++; 54 | print FH_L ">read_$j/1\n"; 55 | print FH_L "$up\n"; 56 | print FH_R ">read_$j/2\n"; 57 | print FH_R "$down\n"; 58 | } 59 | } 60 | close FH_L; 61 | close FH_R; 62 | ``` 63 | 64 | 并不复杂，就十几行代码而已，而且我已经写好了，即使不会写，保证看懂也行，即使看不懂，知道这个代码的用法也行，反正下载地址也给出了。 https://github.com/jmzeng1314/my-perl/blob/master/2.chrX-chrY/simulate.pl 65 | 66 | 整个流程得到的文件如下： 67 | 68 | ``` 69 | 985 Jan 26 2017 calling.sh 70 | 152M Mar 21 2009 chrX.fa 71 | 399 Jan 25 2017 chrX.fa.amb 72 | 44 Jan 25 2017 chrX.fa.ann 73 | 149M Jan 25 2017 chrX.fa.bwt 74 | 23 Jan 26 2017 chrX.fa.fai 75 | 38M Jan 25 2017 chrX.fa.pac 76 | 75M Jan 25 2017 chrX.fa.sa 77 | 58M Mar 21 2009 chrY.fa 78 | 209M Jan 25 2017 read1.fa 79 | 209M Jan 25 2017 read2.fa 80 | 172M Jan 25 2017 read.bam 81 | 3.3M Jan 26 2017 read.bcftools.vcf.gz 82 | 50M Jan 26 2017 read.filter.rmdup.bam 83 | 225K Jan 26 2017 read.filter.rmdup.bam.bai 84 | 668M Jan 25 2017 read.sam 85 | 137M Jan 26 2017 read.sorted.bam 86 | 209K Jan 26 2017 read.sorted.bam.bai 87 | 429 Jan 25 2017 samtools.stat.out 88 | 479 Jan 25 2017 tmp.pl 89 | 409 Jan 25 2017 tmp.sh 90 | ``` 91 | 92 | 93 | 94 | -------------------------------------------------------------------------------- /2.chrX-chrY/simulate.pl: -------------------------------------------------------------------------------- 1 | ############################################# 2 | #Author: Jianming Zeng 3 | #email: jmzeng1314@163.com 4 | #Creat Time: 2017-10-12 11:32:45 5 | #URL1: http://www.bio-info-trainee.com/ 6 | #URL2: https://github.com/jmzeng1314 7 | ### CAFS-->SUSTC-->LCRDC-->university of MACAU 8 | ############################################# 9 | #use strict; 10 | #use warnings; 11 | 12 | my $usage=<read.sam 28 | samtools view -bS read.sam >read.bam 29 | samtools flagstat read.bam 30 | samtools sort -@ 5 -o read.sorted.bam read.bam 31 | samtools view -h -F4 -q 5 read.sorted.bam |samtools view -bS |samtools rmdup - read.filter.rmdup.bam 32 | samtools index read.filter.rmdup.bam 33 | samtools mpileup -ugf ~/tmp/chrX_Y/hg19/chrX.fa read.filter.rmdup.bam |bcftools call -vmO z -o read.bcftools.vcf.gz 34 | ################################################################## 35 | USAGE 36 | 37 | die $usage if @ARGV ne 1; 38 | while(<>){ 39 | chomp; 40 | $chrY.=uc $_; 41 | } 42 | $j=0; 43 | open FH_L,">read1.fa"; 44 | open FH_R,">read2.fa"; 45 | foreach (1..4){ 46 | for ($i=600;$i<(length($chrY)-600);$i = $i+50+int(rand(10))){ 47 | $up = substr($chrY,$i,100); 48 | $down=substr($chrY,$i+400,100); 49 | next unless $up=~/[ATCG]/; 50 | next unless $down=~/[ATCG]/; 51 | $down=reverse $down; 52 | $down=~tr/ATCG/TAGC/; 53 | $j++; 54 | print FH_L ">read_$j/1\n"; 55 | print FH_L "$up\n"; 56 | print FH_R ">read_$j/2\n"; 57 | print FH_R "$down\n"; 58 | } 59 | } 60 | close FH_L; 61 | close FH_R; 62 | -------------------------------------------------------------------------------- /2.chrX-chrY/snp-calling.sh: -------------------------------------------------------------------------------- 1 | mkdir -p ~/tmp/chrX_Y/hg19/ 2 | cd ~/tmp/chrX_Y/hg19/ 3 | #conda install -c bioconda bwa 4 | #conda install -c bioconda samtools 5 | wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/chrX.fa.gz; 6 | wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/chrY.fa.gz; 7 | gunzip chrX.fa.gz 8 | gunzip chrY.fa.gz 9 | wget https://github.com/jmzeng1314/my-perl/blob/master/2.chrX-chrY/simulate.pl 10 | bwa index chrX.faperl simulate.pl chrY.fa 11 | bwa mem -t 5 -M chrX.fa read*.fa >read.sam 12 | samtools view -bS read.sam >read.bam 13 | samtools flagstat read.bam 14 | samtools sort -@ 5 -o read.sorted.bam read.bam 15 | samtools view -h -F4 -q 5 read.sorted.bam |samtools view -bS |samtools rmdup - read.filter.rmdup.bam 16 | samtools index read.filter.rmdup.bam 17 | samtools mpileup -ugf ~/tmp/chrX_Y/hg19/chrX.fa read.filter.rmdup.bam |bcftools call -vmO z -o read.bcftools.vcf.gz 18 | -------------------------------------------------------------------------------- /3.draw_gene_structure/ANXA1-gene-structure.jpg: -------------------------------------------------------------------------------- https://raw.githubusercontent.com/jmzeng1314/my-perl/c865748b97d4565144cbd032669b45fa229d0855/3.draw_gene_structure/ANXA1-gene-structure.jpg -------------------------------------------------------------------------------- /3.draw_gene_structure/step1.gtf2mysql_format.pl: -------------------------------------------------------------------------------- 1 | ###usage::perl gtf2mysql_format.pl gencode.v7.annotation_goodContig.gtf >hg19_gtf2mysql 2 | ###http://www.broadinstitute.org/cancer/cga/rnaseqc_download 3 | while(<>){ 4 | s/\"//g; 5 | undef %h; 6 | next if /^#/; 7 | my($chr,$source,$record,$start,$end,undef,$strand,undef,$info)=split/\t/; 8 | foreach (split/;/,$info){ 9 | my($tag,$value)=split; 10 | $h{$tag}=$value; 11 | } 12 | print join("\t",$h{'gene_name'},$h{'transcript_name'},$record,$chr,$start,$end,$source,$strand, 13 | $h{'gene_id'},$h{'transcript_id'},$h{'gene_status'},$h{'gene_type'},$h{'transcript_type'},$h{'transcript_status'} 14 | )."\n"; 15 | } -------------------------------------------------------------------------------- /3.draw_gene_structure/step2.create_gtf_table.sql: -------------------------------------------------------------------------------- 1 | --mysql code 2 | use test; 3 | drop table if exists hg19_gtf; 4 | create table hg19_gtf ( 5 | gene_name VARCHAR(30), 6 | transcript_name VARCHAR(30) , 7 | record VARCHAR(15) NOT NULL , 8 | chr VARCHAR(2) NOT NULL , 9 | start INT NOT NULL , 10 | end INT NOT NULL , 11 | source VARCHAR(10) NOT NULL , 12 | strand VARCHAR(1) NOT NULL , 13 | gene_id VARCHAR(30) NOT NULL , 14 | transcript_id VARCHAR(30) NOT NULL , 15 | gene_status VARCHAR(30) , 16 | gene_type VARCHAR(30) , 17 | transcript_type VARCHAR(30) , 18 | transcript_status VARCHAR(30) 19 | ); 20 | 21 | --select * from hg19_gtf limit 100; 22 | --select * from hg19_gtf where gene_name='DMD'; 23 | --select count(*) from hg19_gtf where gene_name='DMD' and record='start_codon'; --18 start condon 24 | --select count(distinct(transcript_name)) from hg19_gtf where gene_name='DMD' ; --34 transcript 25 | --select count(distinct(transcript_name)) c ,gene_name from hg19_gtf where record='transcript' group by gene_name order by c desc; 26 | 27 | -------------------------------------------------------------------------------- /3.draw_gene_structure/step3.upload_gtf2mysql.R: -------------------------------------------------------------------------------- 1 | ### R code upload a file to mysql table ! : 2 | setwd('D:\\test_analysis\\gtf') 3 | a=read.table("hg19_gtf2mysql",header=F,stringsAsFactors = F) 4 | ## connect to mysql database 5 | suppressMessages(library(RMySQL)) 6 | con <- dbConnect(MySQL(), host="127.0.0.1", port=3306, user="root", password="11111111") 7 | dbSendQuery(con, "USE test") 8 | ## upload file to table hg19_gtf 9 | field='gene_name,transcript_name,record,chr,start,end,source,strand,gene_id,transcript_id,gene_status,gene_type,transcript_type,transcript_status' 10 | names(a)=strsplit(field,",")[[1]] 11 | dbWriteTable(con,"hg19_gtf",a,overwrite =F,append = T, row.names=FALSE,skip = 0) 12 | ## do some statistics 13 | query="select count(distinct(transcript_name)) c ,gene_name from hg19_gtf where record='transcript' group by gene_name order by c desc;" 14 | stat_t_num=dbGetQuery(con,query) 15 | #hist(stat_t_num[,1]) -------------------------------------------------------------------------------- /3.draw_gene_structure/step4.draw_gene_structure_from_mysql.R: -------------------------------------------------------------------------------- 1 | ## R code to get the gene structure information 2 | ## R code to get the gene structure information 3 | ## R code to get the gene structure information 4 | suppressMessages(library(ggplot2)) 5 | suppressMessages(library(RMySQL)) 6 | con <- dbConnect(MySQL(), host="127.0.0.1", port=3306, user="root", password="11111111") 7 | dbSendQuery(con, "USE test") 8 | gene='SOX10' 9 | #gene='DDX11L11' 10 | if (T){ 11 | query=paste("select * from hg19_gtf where gene_type='protein_coding' and gene_name=",shQuote(gene),sep="") 12 | structure=dbGetQuery(con,query) 13 | tmp_min=min(c(structure$start,structure$end)) 14 | structure$new_start=structure$start-tmp_min 15 | structure$new_end=structure$end-tmp_min 16 | tmp_max=max(c(structure$new_start,structure$new_end)) 17 | num_transcripts=nrow(structure[structure$record=='transcript',]) 18 | tmp_color=rainbow(num_transcripts) 19 | x=1:tmp_max;y=rep(num_transcripts,length(x)) 20 | #x=10000:17000;y=rep(num_transcripts,length(x)) 21 | plot(x,y,type = 'n',xlab='',ylab = '',ylim = c(0,num_transcripts+1)) 22 | title(main = gene,sub = paste("chr",tmp$chr,":",tmp$start,"-",tmp$end,sep="")) 23 | j=0; 24 | tmp_legend=c() 25 | for (i in 1:nrow(structure)){ 26 | tmp=structure[i,] 27 | if(tmp$record == 'transcript'){ 28 | j=j+1 29 | tmp_legend=c(tmp_legend,paste("chr",tmp$chr,":",tmp$start,"-",tmp$end,sep="")) 30 | } 31 | if(tmp$record == 'exon') lines(c(tmp$new_start,tmp$new_end),c(j,j),col=tmp_color[j],lwd=4) 32 | } 33 | # legend('topleft',legend=tmp_legend,lty=1,lwd = 4,col = tmp_color); 34 | 35 | } -------------------------------------------------------------------------------- /LICENSE: -------------------------------------------------------------------------------- 1 | GNU GENERAL PUBLIC LICENSE 2 | Version 3, 29 June 2007 3 | 4 | Copyright (C) 2007 Free Software Foundation, Inc. 5 | Everyone is permitted to copy and distribute verbatim copies 6 | of this license document, but changing it is not allowed. 7 | 8 | Preamble 9 | 10 | The GNU General Public License is a free, copyleft license for 11 | software and other kinds of works. 12 | 13 | The licenses for most software and other practical works are designed 14 | to take away your freedom to share and change the works. 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Of course, your program's commands 662 | might be different; for a GUI interface, you would use an "about box". 663 | 664 | You should also get your employer (if you work as a programmer) or school, 665 | if any, to sign a "copyright disclaimer" for the program, if necessary. 666 | For more information on this, and how to apply and follow the GNU GPL, see 667 | . 668 | 669 | The GNU General Public License does not permit incorporating your program 670 | into proprietary programs. If your program is a subroutine library, you 671 | may consider it more useful to permit linking proprietary applications with 672 | the library. If this is what you want to do, use the GNU Lesser General 673 | Public License instead of this License. But first, please read 674 | . 675 | -------------------------------------------------------------------------------- /README.md: -------------------------------------------------------------------------------- 1 | # my-perl 2 | You can find many short but practical PERL scripts about NGS data analysis in current directory ! 3 | # scripts list 4 | * 1.calc_gene_exon_depth.pl 5 | * 2.get_gene_from_chr_pos.pl 6 | * 3.Degenerate_base2original_base.pl 7 | * 4.snp_mutation_context.pl 8 | * 5.stat_snp_subtype.pl 9 | * 6.stat_WES_depth_covarage.pl 10 | 11 | ## 1.draw exon covarage map for each refseq transcript according a gene sysmbol 12 | 13 | --------------------------------------------------------------------------------