Commit 81e44eae authored by Marwan ELKREWI's avatar Marwan ELKREWI
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Delete ZW_and_Wsnps.md

parent 59c7dcff
**We performed a small backcrossing experiment between one _Artemia sinica_ male and one _Artemia sp. Kazakhstan_ female from inbred lines, with further backcrossing of one F1 hybrid female with an _A.sinica_ male. RNA from 10 male and 10 female F2 hybrid offspring was sequenced and analysed together with RNA from grandparents (_Artemia sinica_ male and _Artemia sp. Kazakhstan_ female).**
![Scheme of the samll beckcrossing experiment](images/Backcrossing_experiment.png)
Working directory for RNA processing: `/nfs/scistore03/vicosgrp/ukhaurat/dummyfather_proj/`
Location of raw RNA files for 20 F2 hybrids and grandparents (RNA from grandparents happened to be sequenced twice, thus we have a pair of files for each grandparent)
```
F2_hybrid_female1.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97667_GACGCATCGAGGCTGC.bam
F2_hybrid_female10.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97681_GTTATCCGGCCGAACG.bam
F2_hybrid_female2.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97668_TGCCGTAGCCTCGTAG.bam
F2_hybrid_female3.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97669_AAGGAATCCATAGGCA.bam
F2_hybrid_female4.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97670_TCGAGTAGAGATGAAC.bam
F2_hybrid_female5.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97671_CTCGAATCCCGAGTAT.bam
F2_hybrid_female6.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97677_TGTGCTTCCCTCTGGC.bam
F2_hybrid_female7.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97678_GTATTAAGCCATTGTG.bam
F2_hybrid_female8.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97679_AAGACTTCACTACGGT.bam
F2_hybrid_female9.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97680_GCCTCTTCAAGTGCTA.bam
F2_hybrid_male1.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97662_GAGCAGCGGGCAGATC.bam
F2_hybrid_male10.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97676_CCAATACGGGTATCTT.bam
F2_hybrid_male2.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97663_TGTTGATCCTATGTTA.bam
F2_hybrid_male3.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97664_GTCCTTCGGTTGACGC.bam
F2_hybrid_male4.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97665_CCGGCATCATCTACGA.bam
F2_hybrid_male5.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/H3GHNBGXC_1/H3GHNBGXC_1#97666_CTTCGTAGCTCGACAG.bam
F2_hybrid_male6.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97672_CGCCGATTAATATTGA.bam
F2_hybrid_male7.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97673_TCGGCGAAGTATACCG.bam
F2_hybrid_male8.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97674_GAGGCCAGGATCCAAC.bam
F2_hybrid_male9.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97675_ATAAGTTCAGATACGC.bam
Gk1.1_kaz_female.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97661_ATGTAGACTTCTTGAA.bam
Gk1.2_kaz_female.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97944_TCAATCAGGACACACT.bam
Gs1.1_sinica_male.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97660_TAACGATTAATAACGT.bam
Gs1.2_sinica_male.bam -> /nfs/scistore03/vicosgrp/ukhaurat/data/HNMWGBGXB_1/HNMWGBGXB_1#97943_CCAGATTCTGTCCACG.bam
```
Links to raw bam files with RNA: `/nfs/scistore03/vicosgrp/ukhaurat/data/links_to_raw_F2hybridsRNA/`
## 1. Transform raw bam files into fasta
```
module load picard
cd /nfs/scistore03/vicosgrp/ukhaurat/proj
#for all the files:
java -jar $PICARD SamToFastq I=F2_hybrid_male2.bam F=F2_hybrid_male2_1.fq F2=F2_hybrid_male2_2.fq
```
`/nfs/scistore03/vicosgrp/ukhaurat/dummyfather_proj/rawfq`.
## 2. Trim raw RNA
```
module load trimmomatic
module load java
for f in /nfs/scistore03/vicosgrp/ukhaurat/dummyfather_proj/*_1.fq
do
base=$(basename ${f%%_*})
srun java -jar /nfs/scistore03/vicosgrp/Bioinformatics_2018/Beatriz/0-Software/Trimmomatic-0.36/trimmomatic-0.36.jar PE -threads 8 ${base}_1.fq ${base}_2.fq ${base}_1.trim.fq ${base}_1un.trim.fq ${base}_2.trim.fq ${base}_2un.trim.fq ILLUMINACLIP:/nfs/scistore03/vicosgrp/Bioinformatics_2018/Beatriz/0-Software/Trimmomatic-0.36/adapters/TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:22 MINLEN:36 LEADING:3 TRAILING:3
done
```
Trimmed RNA reads from 20 hybrids and Kazakhstan and sinica grandparents: `/nfs/scistore03/vicosgrp/ukhaurat/dummyfather_proj/trimmed`.
## 3. Map 20 hybrids RNA and their grandparents to _A.sinica_ reference genome.
Working directory: `/nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/`.
Genome: `/nfs/scistore03/vicosgrp/melkrewi/Project_confirm_genome_assembly/CHRR_integrated.fa`.
```
star_2.0.sh
module load star
cd /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/
STAR --runMode genomeGenerate --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --genomeFastaFiles /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/CHRR_integrated.fa --runThreadN 10 --genomeChrBinNbits 15
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female10_1.trim.fq F2hybrid.female10_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female10_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female9_1.trim.fq F2hybrid.female9_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female9_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female8_1.trim.fq F2hybrid.female8_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female8_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female7_1.trim.fq F2hybrid.female7_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female7_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female6_1.trim.fq F2hybrid.female6_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female6_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female5_1.trim.fq F2hybrid.female5_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female5_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female4_1.trim.fq F2hybrid.female4_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female4_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female3_1.trim.fq F2hybrid.female3_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female3_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female2_1.trim.fq F2hybrid.female2_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female2_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.female1_1.trim.fq F2hybrid.female1_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.female1_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male1_1.trim.fq F2hybrid.male1_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male1_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male2_1.trim.fq F2hybrid.male2_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male2_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male3_1.trim.fq F2hybrid.male3_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male3_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male4_1.trim.fq F2hybrid.male4_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male4_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male5_1.trim.fq F2hybrid.male5_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male5_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male6_1.trim.fq F2hybrid.male6_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male6_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male7_1.trim.fq F2hybrid.male7_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male7_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male8_1.trim.fq F2hybrid.male8_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male8_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male9_1.trim.fq F2hybrid.male9_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male9_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn F2hybrid.male10_1.trim.fq F2hybrid.male10_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/F2hybrid.male10_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn Gk1.kaz.female.v1_1.trim.fq Gk1.kaz.female.v1_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/Gk1.kaz.female.v1_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn Gk1.kaz.female.v2_1.trim.fq Gk1.kaz.female.v2_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/Gk1.kaz.female.v2_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn Gs1.sinica.male.v1_1.trim.fq Gs1.sinica.male.v1_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/Gs1.sinica.male.v1_ --runThreadN 10
STAR --genomeDir /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/index/ --readFilesIn Gs1.sinica.male.v2_2.trim.fq Gs1.sinica.male.v2_2.trim.fq --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/Gs1.sinica.male.v2_ --runThreadN 10
```
#### Mapping statistics
[the google spreadsheet with mapping statistics](https://docs.google.com/spreadsheets/d/1d0LvyZwdyg2QIk675OdFl-pwKgNlR3SULDHPWMaVAPQ/edit?usp=sharing)
## 4. Merge v1 and v2 bams for grandparents
```
i merged bams for kazakhstan and sinica grandparents (made 1 out of two):
java -jar $PICARD MergeSamFiles -I Gk1.kaz.female.v1_Aligned.sortedByCoord.out.bam -I Gk1.kaz.female.v2_Aligned.sortedByCoord.out.bam -O Gk1.kaz.female.both_Aligned.sortedByCoord.out.bam
java -jar $PICARD MergeSamFiles -I Gs1.sinica.male.v1_Aligned.sortedByCoord.out.bam -I Gs1.sinica.male.v2_Aligned.sortedByCoord.out.bam -O Gs1.sinica.male.both_Aligned.sortedByCoord.out.bam
```
## 5. Create a vcf file
I created a vcf file for 20 F2 hybrids and merged bams for grandparents.
the vcf is filtered:
* for minimum and maximum coverage
* for minimum quality
* indels and multi-allelic sites were removed
```
module load samtools
module load bcftools
module load vcftools
cd /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/
#Call SNPs from the BAM alignments
srun bcftools mpileup -a AD,DP,SP -Ou -f CHRR_integrated.fa Gk1.kaz.female.both_Aligned.sortedByCoord.out.bam Gs1.sinica.male.both_Aligned.sortedByCoord.out.bam F2hybrid.female1_Aligned.sortedByCoord.out.bam F2hybrid.female2_Aligned.sortedByCoord.out.bam F2hybrid.female3_Aligned.sortedByCoord.out.bam F2hybrid.female4_Aligned.sortedByCoord.out.bam F2hybrid.female5_Aligned.sortedByCoord.out.bam F2hybrid.female6_Aligned.sortedByCoord.out.bam F2hybrid.female7_Aligned.sortedByCoord.out.bam F2hybrid.female8_Aligned.sortedByCoord.out.bam F2hybrid.female9_Aligned.sortedByCoord.out.bam F2hybrid.female10_Aligned.sortedByCoord.out.bam F2hybrid.male1_Aligned.sortedByCoord.out.bam F2hybrid.male2_Aligned.sortedByCoord.out.bam F2hybrid.male3_Aligned.sortedByCoord.out.bam F2hybrid.male4_Aligned.sortedByCoord.out.bam F2hybrid.male5_Aligned.sortedByCoord.out.bam F2hybrid.male6_Aligned.sortedByCoord.out.bam F2hybrid.male7_Aligned.sortedByCoord.out.bam F2hybrid.male8_Aligned.sortedByCoord.out.bam F2hybrid.male9_Aligned.sortedByCoord.out.bam F2hybrid.male10_Aligned.sortedByCoord.out.bam | bcftools call -v -f GQ,GP -mO z -o head_Gk1_Gs1_f_m.vcf.gz
#filter for quality and coverage
srun vcftools --gzvcf head_Gk1_Gs1_f_m.vcf.gz --remove-indels --maf 0.1 --max-missing 0.9 --minQ 30 --min-meanDP 10 --max-meanDP 100 --minDP 10 --maxDP 100 --recode --stdout > head_Gk1_Gs1_f_m_filtered.vcf
#Filter 2: remove multiallelic
bcftools view --max-alleles 2 --exclude-types indels head_Gk1_Gs1_f_m_filtered.vcf > head_Gk1_Gs1_f_m_filtered2.vcf
```
## 6. Filtering the VCF file and detecting sex-linked scaffolds
- simplify the vcf.
To simplify it so 0/0 is replaced with 0, 0/1 with 1, and 1/1 with 2.
```
cat head_Gk1_Gs1_f_m_filtered2.vcf | grep -v '^##' | perl -pi -e 's/:.*?\t/\t/gi' | perl -pi -e 's/:.*//gi' | perl -pi -e 's/\t/ /gi' | perl -pi -e 's/0\/0/0/gi' | perl -pi -e 's/1\/1/2/gi' | perl -pi -e 's/0\/1/1/gi' | perl -pi -e 's/\.\/\./NA/gi' > head_Gk1_Gs1_f_m_filtered2.vcf_simple
```
- search for W- and ZW-snps.
We want to know which of our SNPs are sex-linked. Here are the possible chromosomal assignments:
* class "0" is autosomal (it was not alternatively fixed in males and females)
* class "1" is a W-SNP (it was found in all the F2 females and none of the males, and it was heterozygous in the inbread grandmother)
* class "2" is a ZW-SNP (it was found in all the F2 females and none of the males, but it was homozygous in the ZW grandmother)
* class "3" is Z-specific (it was homozygous in grandmother and not passed on to F2) [NB: this is not found in the data!]
To run our script, we will use:
* The simplified VCF file head_Gk1_Gs1_f_m_filtered2.vcf_simple
* The script [FindZWSNPs_v2.pl](FindZWSNPs_v2.pl) to filter the vcf
The columns for females, males and grandparents must be specified in the script (first individual = 0).
I changed the original FindZWSNPs_v2.pl into /nfs/scistore03/vicosgrp/ukhaurat/full_assembly_analysis/FindZWSNPs_v2_changed.pl
```
@female_columns = (2, 3, 4, 5, 6, 7, 8, 9, 10, 11);
@male_columns = (12, 13, 14, 15, 16, 17, 18, 19, 20, 21);
$grannycolumn = 0;
$granpacolumn = 1;
```
[FindZWSNPs_v2_.pl](FindZWSNPs_v2.pl) does two things:
1. it filters out SNPs:
* that are 0 or NA in grandmother
* that are 1, 2 or NA in grandfather (since they should have the sinica allele)
* that are 2 or NA in the F2
* that are 1 in fewer than 4 or more than 15 F2 individuals
2. It classifies SNPs into autosomal, W-linked, ZW or Z-specific (see above).
```
perl FindZWSNPs_v2_changed.pl head_Gk1_Gs1_f_m_filtered2.vcf_simple
```
This produces the file head_Gk1_Gs1_f_m_filtered2.vcf_simple.ZWassign, which has the following columns (separated by spaces, I hate tabs now):
* scaffold
* position
* grandmother GT (1 = het, 2 = hom)
* number of heterozygous F2 females
* number of heterozygous F2 males
* chromosomal assignment (autosome "0", W chrom "1", sex chromosomes "2")
We can already have a quick look at how many SNPs get classified as W, ZW, or Z-specific:
```
0 [autosome] 54841
1 [W] 181
2 [ZW] 820
```
Extract the lists of W- and ZW- SNPs
```
cat head_Gk1_Gs1_f_m_filtered2.vcf_simple.ZWassign | awk '($6==1)' > w_snps_perl_script_full_assembly.txt
cat head_Gk1_Gs1_f_m_filtered2.vcf_simple.ZWassign | awk '($6==2)' > zw_snps_perl_script_full_assembly.txt
```
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