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workflow.sh
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# Based on DNA-sequencing workflow from https://github.com/maciej-jonczyk/network-ala/blob/main/map-pl.sh
# Except snpEff all software versions are as in above link
# Individual steps used different number of cores as analysis has been done on different computers
# Sequencing data has been provided with QC in FastQC - no sample require trimming
# 1. Mapping with read-group idntification (all files have the same SM field - it was later changed to allow separate analysis of samples)
# 2024_02_28.map level
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:16_p1\tLB:16_p1\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.GTAACATC+NATCGCTG" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/16_p1_S60_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/16_p1_S60_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 16_p1.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:16_p2\tLB:16_p2\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.CAGCGGTA+NCAAGTCA" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/16_p2_S65_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/16_p2_S65_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 16_p2.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:16_p3\tLB:16_p3\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.GCCTAGCC+NAGACCAA" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/16_p3_S70_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/16_p3_S70_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 16_p3.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:18_p1\tLB:18_p1\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.AGGTAAGG+NAATCGTG" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/18_p1_S61_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/18_p1_S61_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 18_p1.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:18_p2\tLB:18_p2\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.CGCCTTCC+NAGTCCGC" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/18_p2_S66_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/18_p2_S66_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 18_p2.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:18_p3\tLB:18_p3\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.GACCAGGA+NTAGCCAG" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/18_p3_S71_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/18_p3_S71_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 18_p3.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:19_p1\tLB:19_p1\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.ACAGGTAT+NTGGTGGC" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/19_p1_S62_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/19_p1_S62_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 19_p1.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:19_p2\tLB:19_p2\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.CAATAGTC+NTGAGCTG" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/19_p2_S67_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/19_p2_S67_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 19_p2.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:19_p3\tLB:19_p3\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.CAGCAAGG+NTTAGAGG" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/19_p3_S72_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/19_p3_S72_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 19_p3.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:20_p1\tLB:20_p1\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.AATGTTCT+NGGTGACT" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/20_p1_S63_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/20_p1_S63_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 20_p1.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:20_p2\tLB:20_p2\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.ATTATCAA+NAGAGTCG" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/20_p2_S68_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/20_p2_S68_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 20_p2.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:20_p3\tLB:20_p3\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.CGTTACCA+NAGCGTTC" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/20_p3_S73_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/20_p3_S73_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 20_p3.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:22_p1\tLB:22_p1\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.TCTGCAAG+NAGGTGAA" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/22_p1_S64_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/22_p1_S64_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 22_p1.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:22_p2\tLB:22_p2\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.CCAACATT+NAGAGAGC" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/22_p2_S69_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/22_p2_S69_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 22_p2.bam
~/bin/bwa-mem2-2.2.1_x64-linux/bwa-mem2 mem -t8 -T30 \
-R "@RG\tID:22_p3\tLB:22_p3\tPL:illumina\tSM:s68911\tPU:H3N5LDSXC.1.TCCAGCAA+NGCCAATT" \
/media/mj/Seagate\ Backup\ Plus\ Drive/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa \
/media/mj/Expansion/2024_02_28/raw_data/22_p3_S74_R1_001.fastq.gz \
/media/mj/Expansion/2024_02_28/raw_data/22_p3_S74_R2_001.fastq.gz \
| ~/bin/samtools-1.17/samtools view -bo 22_p3.bam
# 2. bamQC, split to three pararell analyses
# new form of loop was used which added prefix to input name to create output file
for file in *1.bam; do \
~/bin/BamQC-master/bin/bamqc \
$file \
--threads 4 \
-f /home/mj/NAMv5/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.55.chr.gtf \
-o bamqc-raw; done &
for file in *2.bam; do \
~/bin/BamQC-master/bin/bamqc \
$file \
--threads 4 \
-f /home/mj/NAMv5/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.55.chr.gtf \
-o bamqc-raw; done &
for file in *3.bam; do \
~/bin/BamQC-master/bin/bamqc \
$file \
--threads 4 \
-f /home/mj/NAMv5/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.55.chr.gtf \
-o bamqc-raw; done
# 3. Quality filtering
# 2024_02_28.map level
for i in *.bam; do \
~/bin/samtools-1.20/samtools view -@4 -bq30 \
$i \
-o q30/q30.$i; done
# 4. bamQC
# q30 level
for file in *1.bam; do \
~/bin/BamQC-master/bin/bamqc \
$file \
--threads 4 \
-f /home/mj/NAMv5/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.55.chr.gtf \
-o ../bamqc-q30; done &
for file in *2.bam; do \
~/bin/BamQC-master/bin/bamqc \
$file \
--threads 4 \
-f /home/mj/NAMv5/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.55.chr.gtf \
-o ../bamqc-q30; done &
for file in *3.bam; do \
~/bin/BamQC-master/bin/bamqc \
$file \
--threads 4 \
-f /home/mj/NAMv5/Zea_mays.Zm-B73-REFERENCE-NAM-5.0.55.chr.gtf \
-o ../bamqc-q30; done
# 5. Sort by name, required for deduplication
# q30 level
for file in *.bam; do \
java -Xmx100g -jar ~/bin/picard.jar SortSam \
-I $file \
-O name-srt/sn-$file \
--SORT_ORDER queryname; done
# 6. Deduplication
# name-srt level
# read-regex check
samtools view <FILE>.bam | head | grep -E '([a-zA-Z0-9]+):([0-9]+):([a-zA-Z0-9]+):([0-9]+):([0-9]+):([0-9]+):([0-9]+)'
# deduplication itself
cd name-srt
for file in *.bam; do \
java -Xmx100g -jar ~/bin/picard.jar MarkDuplicates \
-I $file \
-O ../dedup/dedup-$file \
-M ../dedup/metrics-dedup-$file \
--READ_NAME_REGEX '([a-zA-Z0-9]+):([0-9]+):([a-zA-Z0-9]+):([0-9]+):([0-9]+):([0-9]+):([0-9]+)' \
--REMOVE_DUPLICATES true \
--ASSUME_SORT_ORDER queryname \
--OPTICAL_DUPLICATE_PIXEL_DISTANCE 100 \
--TAGGING_POLICY All ; done
# 7. Sort by coordinate
# q30/dedup/ level
for f in *.bam; do \
samtools sort -@24 ${f} -o ../../sort4varcall/srt-${f}
done
# 8. CORRECT read-group SM field (all previous files have bad value)
# sort4varcall level
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.16_p1.bam O=popr-readgroup/16_p1.bam RGID=16_p1 RGLB=16_p1 RGPL=illumina RGPU=H3N5LDSXC.1.GTAACATC+NATCGCTG RGSM=16_p1
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.16_p2.bam O=popr-readgroup/16_p2.bam RGID=16_p2 RGLB=16_p2 RGPL=illumina RGPU=H3N5LDSXC.1.CAGCGGTA+NCAAGTCA RGSM=16_p2
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.16_p3.bam O=popr-readgroup/16_p3.bam RGID=16_p3 RGLB=16_p3 RGPL=illumina RGPU=H3N5LDSXC.1.GCCTAGCC+NAGACCAA RGSM=16_p3
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.18_p1.bam O=popr-readgroup/18_p1.bam RGID=18_p1 RGLB=18_p1 RGPL=illumina RGPU=H3N5LDSXC.1.AGGTAAGG+NAATCGTG RGSM=18_p1
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.18_p2.bam O=popr-readgroup/18_p2.bam RGID=18_p2 RGLB=18_p2 RGPL=illumina RGPU=H3N5LDSXC.1.CGCCTTCC+NAGTCCGC RGSM=18_p2
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.18_p3.bam O=popr-readgroup/18_p3.bam RGID=18_p3 RGLB=18_p3 RGPL=illumina RGPU=H3N5LDSXC.1.GACCAGGA+NTAGCCAG RGSM=18_p3
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.19_p1.bam O=popr-readgroup/19_p1.bam RGID=19_p1 RGLB=19_p1 RGPL=illumina RGPU=H3N5LDSXC.1.ACAGGTAT+NTGGTGGC RGSM=19_p1
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.19_p2.bam O=popr-readgroup/19_p2.bam RGID=19_p2 RGLB=19_p2 RGPL=illumina RGPU=H3N5LDSXC.1.CAATAGTC+NTGAGCTG RGSM=19_p2
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.19_p3.bam O=popr-readgroup/19_p3.bam RGID=19_p3 RGLB=19_p3 RGPL=illumina RGPU=H3N5LDSXC.1.CAGCAAGG+NTTAGAGG RGSM=19_p3
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.20_p1.bam O=popr-readgroup/20_p1.bam RGID=20_p1 RGLB=20_p1 RGPL=illumina RGPU=H3N5LDSXC.1.AATGTTCT+NGGTGACT RGSM=20_p1
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.20_p2.bam O=popr-readgroup/20_p2.bam RGID=20_p2 RGLB=20_p2 RGPL=illumina RGPU=H3N5LDSXC.1.ATTATCAA+NAGAGTCG RGSM=20_p2
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.20_p3.bam O=popr-readgroup/20_p3.bam RGID=20_p3 RGLB=20_p3 RGPL=illumina RGPU=H3N5LDSXC.1.CGTTACCA+NAGCGTTC RGSM=20_p3
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.22_p1.bam O=popr-readgroup/22_p1.bam RGID=22_p1 RGLB=22_p1 RGPL=illumina RGPU=H3N5LDSXC.1.TCTGCAAG+NAGGTGAA RGSM=22_p1
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.22_p2.bam O=popr-readgroup/22_p2.bam RGID=22_p2 RGLB=22_p2 RGPL=illumina RGPU=H3N5LDSXC.1.CCAACATT+NAGAGAGC RGSM=22_p2
java -jar ~/bin/picard.jar AddOrReplaceReadGroups I=srt-dedup-sn-q30.22_p3.bam O=popr-readgroup/22_p3.bam RGID=22_p3 RGLB=22_p3 RGPL=illumina RGPU=H3N5LDSXC.1.TCCAGCAA+NGCCAATT RGSM=22_p3
# 9. Index
cd popr-readgroup
for f in *.bam ; \
do \
samtools index -@24 ${f} ; \
done
# 10. Variant-calling
ulimit -n 20000 # required by freebayes-pararell
/home/mj/bin/freebayes-scripts/freebayes-parallel <(/home/mj/bin/freebayes-scripts/fasta_generate_regions.py \
/media/mj/bb71e88b-2122-487c-a602-a2552c680021/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa.fai 100000) 24 \
-f /media/mj/bb71e88b-2122-487c-a602-a2552c680021/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa 16_p1.bam 16_p2.bam 16_p3.bam 18_p1.bam 18_p2.bam 18_p3.bam 19_p1.bam \
19_p2.bam 19_p3.bam 20_p1.bam 20_p2.bam 20_p3.bam 22_p1.bam 22_p2.bam 22_p3.bam \
/media/mj/17d60f37-45c8-4878-8d94-7e95ff7bbddb/map-pl/sort4varcall/srt-hds1-dedup.bam > ../../varcall-parallel/s68.vcf 2>../../varcall-parallel/info
# 11. Quality-filtering
cd ../../varcall-parallel
vcffilter -f "QUAL > 20 & DP > 5" s68.vcf > s68q20dp5.vcf
# 12. QC
~/bin/bcftools-1.17/bcftools stats -s - s68q20dp5.vcf -F /media/mj/bb71e88b-2122-487c-a602-a2552c680021/NAMv5/Zm-B73-REFERENCE-NAM-5.0.fa > stats20dp5
# multiqc
multiqc .
# plot
~/bin/bcftools-1.17/misc/plot-vcfstats -p plot -s stats20dp5
# 13. Annotation
java -Xmx100G -jar ~/bin/snpEff/snpEff.jar ann -csvStats stat-def.csv -s stat-def.html -c ~/bin/snpEff/snpEff.config Zea_mays -v s68q20dp5.vcf > ann.s68q20dp5.vcf 2>bledy-def
# 14. Multi Bam Summary
# sort4varcall level
# all files
multiBamSummary bins --bamfiles \
srt-dedup-sn-q30.16_p1.bam srt-dedup-sn-q30.16_p2.bam srt-dedup-sn-q30.16_p3.bam srt-dedup-sn-q30.18_p1.bam srt-dedup-sn-q30.18_p2.bam srt-dedup-sn-q30.18_p3.bam srt-dedup-sn-q30.19_p1.bam srt-dedup-sn-q30.19_p2.bam srt-dedup-sn-q30.19_p3.bam srt-dedup-sn-q30.20_p1.bam srt-dedup-sn-q30.20_p2.bam srt-dedup-sn-q30.20_p3.bam srt-dedup-sn-q30.22_p1.bam srt-dedup-sn-q30.22_p2.bam srt-dedup-sn-q30.22_p3.bam /media/mj/17d60f37-45c8-4878-8d94-7e95ff7bbddb/map-pl/sort4varcall/srt-hds1-dedup.bam \
--labels \
16_p1 16_p2 16_p3 18_p1 18_p2 18_p3 19_p1 19_p2 19_p3 20_p1 20_p2 20_p3 22_p1 22_p2 22_p3 hds1 \
-p 24 -o ../pca/s68.npz
# Without old bam (HDS-1 from 2014)
# popr-readgroup level
multiBamSummary bins --bamfiles \
16_p1.bam 16_p2.bam 16_p3.bam 18_p1.bam 18_p2.bam 18_p3.bam 19_p1.bam 19_p2.bam 19_p3.bam 20_p1.bam 20_p2.bam 20_p3.bam 22_p1.bam 22_p2.bam 22_p3.bam \
--labels \
16_p1 16_p2 16_p3 18_p1 18_p2 18_p3 19_p1 19_p2 19_p3 20_p1 20_p2 20_p3 22_p1 22_p2 22_p3 \
-p 24 -o ../../pca/s68-noold.npz
# 15. PCA plots
plotPCA -in s68.npz \
-l \
16_p1 16_p2 16_p3 18_p1 18_p2 18_p3 19_p1 19_p2 19_p3 20_p1 20_p2 20_p3 22_p1 22_p2 22_p3 hds1 \
--colors \
'#66ff66' '#33cc33' '#009933' '#6699ff' '#3366ff' '#3333ff' '#ff66ff' '#ff00ff' '#cc00cc' '#ff6666' '#ff5050' '#cc0000' '#ffff00' '#cc9900' '#996633' '#000000' \
--markers \
'o' \
--transpose --plotHeight 20 --plotWidth 20 -o s68.svg
plotPCA -in s68-noold.npz \
-l \
16_p1 16_p2 16_p3 18_p1 18_p2 18_p3 19_p1 19_p2 19_p3 20_p1 20_p2 20_p3 22_p1 22_p2 22_p3 \
--colors \
'#33cc33' '#33cc33' '#33cc33' '#3366ff' '#3366ff' '#3366ff' '#ff00ff' '#ff00ff' '#ff00ff' '#ff5050' '#ff5050' '#ff5050' '#cccc00' '#cccc00' '#cccc00' \
--markers \
'o' 'x' 'v' \
--transpose --plotHeight 20 --plotWidth 20 -o s68noold.svg