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12 days ago
Nesma
•
0
**Hello everyone, I've been trying to generate vcf files for my samples. I did a single sample as a start just to see if everything is going well. could you please take a look at the output and let me know if you see anything strange? (I'm a beginner and this my first time generating a vcf file) Is it normal that the quality score is the same for all sites? This is the output of command:
head -n 50 1_16_variants.vcf
NC_007795.1 62 . G A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 66 . G A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 89 . C T 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 93 . C T 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 137 . A G 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 152 . T A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 199 . T C 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 205 . G C 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 210 . C T 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 358 . T A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 393 . A G 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 411 . A G 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 467 . C T 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 621 . T C 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 735 . T A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 805 . A G 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 903 . C T 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 915 . T C 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 1194 . C T 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 1197 . G A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 1281 . A G 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 1308 . C T 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 1320 . A G 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 1689 . G A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 1917 . C T 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 1924 . G A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 2011 . A G 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 2269 . G A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 2593 . G A 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
NC_007795.1 2605 . A G 30.4183 . DP=1;SGB=-0.379885;MQ0F=0;AC=2;AN=2;DP4=0,0,1,0;MQ=60 GT:PL:DP 1/1:60,3,0:1
And these are the commands I used:
bwa index GCF_000013425.1_ASM1342v1_genomic.fna
bwa mem GCF_000013425.1_ASM1342v1_genomic.fna 1-16_S7_L001.fasta | samtools view -O BAM - | samtools sort -T temp -O bam -o 1-16.sorted.bam –
samtools index 1-16.sorted.bam
picard MarkDuplicates I=1-16.sorted.bam O=1-16.markdup.bam M=1-16.metrics.txt
command proceeded succefully but this message appeared:
You may need to specify a READ_NAME_REGEX in order to correctly identify optical duplicates
samtools index 1-16.markdup.bam
samtools stats 1-16.markdup.bam > 1-16.markdup.stats
plot-bamstats -p 1-16_plot/ 1-16.markdup.stats
the following error occurs:
set terminal png size 600,400 truecolor
"1-16_plot/quals.gp", line 2: unknown or ambiguous terminal type; type just 'set terminal' for a list
The command exited with non-zero status 256:
gnuplot 1-16_plot/quals.gp
at /home/manager/miniconda2/bin/plot-bamstats line 51.
main::error("The command exited with non-zero status 256:\x{a}\x{9}gnuplot 1-16_pl"...) called at /home/manager/miniconda2/bin/plot-bamstats line 336
main::plot("1-16_plot/quals.gp") called at /home/manager/miniconda2/bin/plot-bamstats line 682
main::plot_qualities(HASH(0x5621b0a46cf8)) called at /home/manager/miniconda2/bin/plot-bamstats line 33
samtools mpileup -t DP -Bug -m 4 -f GCF_000013425.1_ASM1342v1_genomic.fna 1-16.markdup.bam > 1-16_variants.bcf
bcftools call -mv -O v -o 1-16_variants.vcf 1-16_variants.bcf
command proceeded but this message appeared:
Note: none of --samples-file, --ploidy or --ploidy-file given, assuming all sites are diploid
Apologies for the inconvenience.
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I didn't know I'm sorry, I think I fixed it
the depth DP at all your sites is only '1'