HP : Heteroplasmy Pipeline

SYSTEM PREREQUISITES

OS:                UNIX/LINUX 
SOFTWARE PACKAGES: git,wget,tar,unzip,make,autoconf,gcc,java,perl,python

PIPELINE PREREQUISITES

SOFTWARE PACKAGES: bwa,samtools,bedtools,fastp,samblaster,bcftools,htslib
JAVA JARS:         gatk,mutserve,haplogrep,haplocheck
HUMAN ASSEMBLY:    hs38DH

INSTALL

DOWNLOAD PIPELINE

$ git clone https://github.com/dpuiu/HP.git

SETUP ENVIRONMENT

$ export HP_SDIR=`pwd`/HP/scripts/  # set script directory variable (add to ~/.bashrc)

INSTALL SYSTEM PREREQUISITES

$ $HP_SDIR/install_sysprerequisites.sh		        # perl,pthon,java,wget ...

INSTALL PREREQUISITES ; CHECK INSTALL

$ $HP_SDIR/install_prerequisites.sh  			# bwa,samtools,bedtools ...
  
$ $HP_SDIR/checkInstall.sh
# if successfull => "Success message!"

$ cat checkInstall.log					# contains version/path info

USAGE

SETUP ENVIRONMENT

# go to your working directory 

$ cp -i $HP_SDIR/init.sh .                                   # copy init to working dir.

$ nano init.sh                                               # check/edit local init file
    ...
   export HP_ADIR=$PWD/bams/                                 # alignment dir
   export HP_ODIR=$PWD/out/                                  # output dir  
   export HP_IN=$PWD/in.txt                                  # input file
   export HP_L=                                              # number of reads to subsample
                                                             #   empty if all reads should be used
   find $HP_ADIR/ -name "*.bam" -o -name "*.cram" | \
    $HP_SDIR/ls2in.pl -out $HP_ODIR | sort -V > $HP_IN       # generate input file; 3 column
  ...                                                        # tab delimited, can be edited

$ . ./init.sh                                                # source init file 

$ printenv | grep HP_ | sort                                 # optional ; check HP_ variables 
$ nano $HP_IN                                                # optional ; check input file

RUN PIPELINE

$ $HP_SDIR/run.sh > run.all.sh                               # create command file in working directory
$ bash ./run.all.sh                                          # execute command file

RE-RUN PIPELINE (optional)

$ nano init.sh                                               # update parameters
$ $HP_SDIR/run.sh > run.all.sh                               # recreate command file

$ bash ./run.all.sh

OUTPUT

under $HP_ODIR:

TAB/SUMMARY/VCF/FASTA Files: 

count.tab                                                    # reads  & mtDNA-CN counts
cvg.tab                                                      # coverage stats

1st ITERATION

{mutect2,mutserve}.{03,05,10}.{concat,merge[.sitesOnly]}.vcf # SNVs; 3,5,10% heteroplasmy thold
{mutect2,mutserve}.{03,05,10}.tab                            # SNV counts
{mutect2,mutserve}.{03,05,10}.summary                        # SNV count summaries

{mutect2,mutserve}.fa                                        # consensus sequence
{mutect2,mutserve}.haplogroup[1].tab                         # haplogroup
{mutect2,mutserve}.haplocheck.tab                            # contamination screen

2nd ITERATION

mutect2.mutect2.{03,05,10}.{concat,merge[.sitesOnly]}.vcf	
mutect2.mutect2.{03,05,10}.tab

EXAMPLE 1 : Usage

$ $HP_SDIR/run.sh

EXAMPLE 2 : 3 simulated datasets

input file

$ head $HP_IN
  #sampleName     inputFile          outputPath/prefix
  chrM.A          bams/chrM.A.bam    out/chrM.A/chrM.A
  chrM.B          bams/chrM.B.bam    out/chrM.B/chrM.B
  chrM.C          bams/chrM.C.bam    out/chrM.C/chrM.C
  ...

read counts and mtDNA-CN(M)

 $ head $HP_ODIR/count.tab
   Run	 all        mapped     chrM    M
   chrM.A    851537886  848029490  396766  181.7
   chrM.B    884383716  882213718  506597  223.01
   chrM.C    786560467  785208588  503241  248.9
   ...

directory structure

$ ls $HP_ADIR/*
  bams/chrM.A.bam
  bams/chrM.A.bam.bai
  bams/chrM.A.idxstats
  bams/chrM.A.count
 ...

after running the pipeline

output directories : 1/sample

$ ls out/
  chrM.A/
  chrM.B/
  chrM.C/ 
  ...

vcf files : concat & merge

# 1st iteration : homoplasmies(AF=1) and heteroplasmies(AF<1)
$ cat mutect2.03.concat.vcf  
  ...
  ##INFO=<ID=SM,Number=1,Type=String,Description="Sample">
  ##INFO=<ID=HP,Number=0,Type=Flag,Description="Homoloplymer">
  ##INFO=<ID=HS,Number=0,Type=Flag,Description="Hot spot">
  ##INFO=<ID=CDS,Number=0,Type=Flag,Description="coding region">
  ##INFO=<ID=RNR,Number=0,Type=Flag,Description="rRNA">
  ##INFO=<ID=TRN,Number=0,Type=Flag,Description="tRNA">
  ##INFO=<ID=DLOOP,Number=0,Type=Flag,Description="D-loop">
  ##INFO=<ID=HG,Number=1,Type=String,Description="haplogroup">
  ##INFO=<ID=NUMT,Number=0,Type=Flag,Description="NUMT like">
  ##INFO=<ID=HV,Number=0,Type=Flag,Description="Hypervariable">
  ##INFO=<ID=SM,Number=1,Type=String,Description="Sample">
  ##INFO=<ID=CR,Number=1,Type=String,Description="CADD Raw score">
  ##INFO=<ID=CP,Number=1,Type=String,Description="CADD PHREDD score">
  ...
  ##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
  ##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Read Depth">
  ##FORMAT=<ID=AF,Number=A,Type=Float,Description="Allele Frequency">
  ...
  #CHROM  POS  ID  REF  ALT  QUAL  FILTER                        INFO                                          FORMAT    SAMPLE
  chrM    64   .   C    T    .     clustered_events;haplotype    SM=chrM.A;HV;DLOOP;CR=1.140555;CP=11.43       GT:DP:AF  0|1:67:1
  chrM    73   .   A    G    .     PASS                          SM=chrM.B;HV;DLOOP;NUMT;CR=1.039800;CP=10.89  GT:DP:AF  0/1:52:1
  chrM    73   .   A    G    .     PASS                          SM=chrM.C;HV;DLOOP;NUMT;CR=1.039800;CP=10.89  GT:DP:AF  0/1:43:1
  chrM    73   .   A    G    .     clustered_events;haplotype    SM=chrM.A;HV;DLOOP;NUMT;CR=1.039800;CP=10.89  GT:DP:AF  0|1:70:1
  chrM    146  .   T    C    .     clustered_events;haplotype    SM=chrM.A;HV;DLOOP;CR=0.949142;CP=10.37       GT:DP:AF  0|1:88:1
  ...
  chrM    374  .   A    G    .     clustered_events              SM=chrM.A;DLOOP;CR=0.998787;CP=10.66          GT:DP:AF  0/1:76:0.139
  chrM    375  .   C    T    .     clustered_events;strand_bias  SM=chrM.B;DLOOP;CR=1.074472;CP=11.08          GT:DP:AF  0/1:65:0.162
  chrM    378  .     C    T    .   clustered_events;strand_bias  SM=chrM.C;DLOOP;CR=1.043995;CP=10.91          GT:DP:AF  0/1:82:0.128
  ...
  chrM    1037 .   A    C    .     PASS                          SM=chrM.B;RNR;CR=0.682748;CP=8.714            GT:DP:AF  0/1:70:0.178
  chrM    1038 .   C    G    .     PASS                          SM=chrM.A;RNR;CR=0.822718;CP=9.603            GT:DP:AF  0/1:70:0.165
  chrM    1042 .   T    A     .    PASS                          SM=chrM.C;RNR;CR=1.104043;CP=11.24            GT:DP:AF  0/1:60:0.24
  ...
$ cat mutect2.03.merge.vcf  
  ...  
  #CHROM  POS  ID  REF  ALT  QUAL  FILTER  INFO                                          FORMAT    chrM.A    chrM.B    chrM.C
  chrM    64   .   C    T    .     .       AC=1;AN=2;HV;DLOOP;CR=1.140555;CP=11.43       GT:DP:AF  0|1:67:1  .:.:.     .:.:.
  chrM    73   .   A    G    .     .       AC=3;AN=6;HV;DLOOP;NUMT;CR=1.039800;CP=10.89  GT:DP:AF  0|1:70:1  0/1:52:1  0/1:43:1
  chrM    146  .   T    C    .     .       AC=1;AN=2;HV;DLOOP;CR=0.949142;CP=10.37       GT:DP:AF  0|1:88:1  .:.:.     .:.:.
  ...

$ cat mutect2.03.merge.sitesOnly.vcf
 ...
  #CHROM  POS  ID  REF  ALT  QUAL  FILTER  INFO
  chrM    64   .   C    T    .     .       AC=1;AN=2;HV;DLOOP;CR=1.140555;CP=11.43
  chrM    73   .   A    G    .     .       AC=3;AN=6;HV;DLOOP;NUMT;CR=1.039800;CP=10.89
  chrM    146  .   T    C    .     .       AC=1;AN=2;HV;DLOOP;CR=0.949142;CP=10.37
  ...

# 2nd iteration ; mostly heteroplasmies(AF<1)
$ cat mutect2.mutect2.03.concat.vcf 
  #CHROM  POS   ID  REF   ALT  QUAL  FILTER  INFO                                  FORMAT    SAMPLE
  chrM    374   .   A     G    .     PASS    SM=chrM.A;DLOOP;CR=0.998787;CP=10.66  GT:DP:AF  0/1:62:0.138
  chrM    375   .   C     T    .     PASS    SM=chrM.B;DLOOP;CR=1.074472;CP=11.08  GT:DP:AF  0/1:48:0.178
  chrM    378   .   C     T    .     PASS    SM=chrM.C;DLOOP;CR=1.043995;CP=10.91  GT:DP:AF  0/1:71:0.121
  ...
  chrM    1037  .   A     C    .     PASS    SM=chrM.B;RNR;CR=0.682748;CP=8.714    GT:DP:AF  0/1:70:0.178
  chrM    1038  .   C     G    .     PASS    SM=chrM.A;RNR;CR=0.822718;CP=9.603    GT:DP:AF  0/1:70:0.165
  chrM    1042  .   T     A    .     PASS    SM=chrM.C;RNR;CR=1.104043;CP=11.24    GT:DP:AF  0/1:60:0.24
  ...

SNV counts

# 1st iteration
$ cat mutect2.03.tab            
  Run     H   h   S   s   I  i  Hp  hp  Sp  sp  Ip  ip  A
  chrM.A  31  43  28  35  3  8  28  43  28  35  0   8   74
  chrM.B  27  43  25  35  2  8  22  41  22  33  0   8   70
  chrM.C  40  42  36  34  4  8  38  41  36  33  2   8   82
  ...

# 2nd iteration ; no homoplamies(H=S=I=0)
$ cat  mutect2.mutect2.03.tab 
  Run     H  h   S  s   I  i  Hp  hp  Sp  sp  Ip  ip  A
  chrM.A  0  43  0  35  0  8  0   43  0   35  0   8   43
  chrM.B  0  43  0  35  0  8  0   41  0   33  0   8   43
  chrM.C  0  42  0  34  0  8  0   41  0   33  0   8   42
  ...

Summaries

# 1st iteration
$ cat mutect2.03.summary  | column -t
  id  count  nonZero  min  max  median  mean   sum
   H   3      3        27   39   31      32.33  97
   h   3      3        42   44   43      43     129
   S   3      3        25   35   28      29.33  88
   s   3      3        34   36   35      35     105
   I   3      3        2    4    3       3      9
   i   3      3        8    8    8       8      24
   Hp  3      3        22   37   28      29     87
   hp  3      3        42   44   43      43     129
   Sp  3      3        22   35   28      28.33  85
   sp  3      3        34   36   35      35     105
   Ip  3      1        0    2    0       0.67   2
   ip  3      3        8    8    8       8      24
   A   3      3        69   82   75      75.33  226

Haplogroups

$ cat mutect2.haplogroup.tab    
  Run                        haplogroup  
  chrM.A                     A2+(64)     
  chrM.B                     B2          
  chrM.C                     C           
  ...

Contamination

$ cat mutect2.haplocheck.tab  | column -t
  Run     ContaminationStatus  ContaminationLevel  Distance  SampleCoverage
  chrM.A  NO                   ND                  14        78
  chrM.B  NO                   ND                  14        79
  chrM.C  NO                   ND                  14        78
  ...

EXAMPLE 3 : CUSTOM FILTERING

using init.sh

$ nano init.sh
  export HP_FNAME="no_qual_haplotype_strand"
  export HP_FRULE="egrep -v \"qual|haplotype|strand\""
# rerun run.sh ...

command line

$ HP_M=mutect2
$ cat $HP_ODIR/$M.00.concat.vcf  | egrep -v 'qual|haplotype|strand'  > \
    $HP_ODIR/$M.no_qual_haplotype_strand.00.concat.vcf
$ $HP_SDIR/snpCount.sh $HP_IN $ODIR $HP_M.no_qual_haplotype_strand 03
$ $HP_SDIR/snpCount.sh $HP_IN $ODIR $HP_M.no_qual_haplotype_strand 05
$ $HP_SDIR/snpCount.sh $HP_IN $ODIR $HP_M.no_qual_haplotype_strand 10

LEGEND

Run                           # Sample name

all                           # number of reads in the sample reads
mapped                        # number of aligned reads in the sample
chrM                          # number of reads aligned to chrM
filter                        # number of chrM used (subsample ~2000x cvg based on name)

Gcvg                          # recomputed genome coverage: Bases/3217346917
Mcvg                          # mitochondrion covearge: chrM*rdLen/16569
M                             # mtDNA copy number ; = 2*Mcvg/Gcvg

haplogroup                    # sample haplogroup

H                             # number of homozygous SNVs (the multiallelic SNVs counted once)
h                             # number of heterozygous SNVs
S                             # number of homozygous SNVs (excluding INDELs)
s                             # number of heterozygous SNVs (excluding INDELs)
I                             # number of homozygous INDELs
i                             # number of heterozygous INDELs
...
?p                            # "p" suffix stands for non homopolimeric
...
A                             # total number of SNVs (H+h)

Name		Name	Last commit message	Last commit date
Latest commit History 281 Commits
RefSeq		RefSeq
examples1		examples1
examples2		examples2
graphviz		graphviz
scripts		scripts
DNAnexus_CLOUD.md		DNAnexus_CLOUD.md
GOOGLE_CLOUD.md		GOOGLE_CLOUD.md
README.md		README.md

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HP : Heteroplasmy Pipeline

SYSTEM PREREQUISITES

PIPELINE PREREQUISITES

INSTALL

DOWNLOAD PIPELINE

SETUP ENVIRONMENT

INSTALL SYSTEM PREREQUISITES

INSTALL PREREQUISITES ; CHECK INSTALL

USAGE

SETUP ENVIRONMENT

RUN PIPELINE

RE-RUN PIPELINE (optional)

OUTPUT

1st ITERATION

2nd ITERATION

EXAMPLE 1 : Usage

EXAMPLE 2 : 3 simulated datasets

input file

read counts and mtDNA-CN(M)

directory structure

after running the pipeline

output directories : 1/sample

vcf files : concat & merge

SNV counts

Summaries

Haplogroups

Contamination

EXAMPLE 3 : CUSTOM FILTERING

using init.sh

command line

LEGEND

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HP : Heteroplasmy Pipeline

SYSTEM PREREQUISITES

PIPELINE PREREQUISITES

INSTALL

DOWNLOAD PIPELINE

SETUP ENVIRONMENT

INSTALL SYSTEM PREREQUISITES

INSTALL PREREQUISITES ; CHECK INSTALL

USAGE

SETUP ENVIRONMENT

RUN PIPELINE

RE-RUN PIPELINE (optional)

OUTPUT

1st ITERATION

2nd ITERATION

EXAMPLE 1 : Usage

EXAMPLE 2 : 3 simulated datasets

input file

read counts and mtDNA-CN(M)

directory structure

after running the pipeline

output directories : 1/sample

vcf files : concat & merge

SNV counts

Summaries

Haplogroups

Contamination

EXAMPLE 3 : CUSTOM FILTERING

using init.sh

command line

LEGEND

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