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II. Genomic Variation Analysis

This chapter contains a manual on variant calling pipeline. Details are described here only on how to conduct such a study from the time the data are downloaded to the time the genetic variant annotation files are received. The biological interpretation of the results is not given here.

Instruction

CLT
IDE

You can run commands below in your terminal.
But if you want to write a convenient to read laboratory journal you can use Jupyter Notebook.
In that case write ! in the beggining of each cell to make it understand bash commands.

To recreate any of the steps of this manual please install:

wget https://github.com/iliapopov17/NGS-Handbook/raw/refs/heads/main/envs/varcall.yaml

conda env create -f varcall.yaml

And of cource do not forget to activate the envinronment!

conda activate varcall

Step 1: Downloading the data

First we create all the directories to store the data

Input

mkdir data/
mkdir data/reference/

Download reference file from NCBI in FASTA format

Input

efetch -db nuccore -id U00096.3 -format fasta > data/reference/EcoliK12MG1655.fa

Download reference file from NCBI in GB format

Input

efetch -db nuccore -id U00096.3 -format gb > data/reference/EcoliK12MG1655.gb

Download sample FASTQ files from SRA via sra-tools

Input

fastq-dump -v --split-3 --gzip SRR17909485 -O data

Output

Preference setting is: Prefer SRA Normalized Format files with full base quality scores if available.
SRR17909485 is an SRA Normalized Format file with full base quality scores.
Read 1251776 spots for SRR17909485
Written 1251776 spots for SRR17909485

Step 2: Check the reads quality

Input

mkdir data/fastqc_results/

Input

fastqc data/SRR17909485_*.fastq.gz -o data/fastqc_results

Output

Results are saved to:
- SRR17909485_1_fastqc.html
- SRR17909485_2_fastqc.html
Please open them in any web browser you use:

Per base sequence quality

Forward read Reverse read

Per sequence quality scores

Forward read Reverse read

Per base sequence content

Forward read Reverse read

Per sequence GC content

Forward read Reverse read

Adapter Content

Forward read Reverse read

The main problem is Per base sequence quality. So we will solve it using trimmomatic.
Details on trimmomatic and QC in the Quality Control chapter.

Step 3: Trim low-quality bases

Input

mkdir data/trimmed/

Input

trimmomatic PE -threads 2 data/SRR17909485_1.fastq.gz data/SRR17909485_2.fastq.gz \
    data/trimmed/SRR17909485_R1.trim.paired.fastq.gz data/trimmed/SRR17909485_R1.trim.unpaired.fastq.gz \
    data/trimmed/SRR17909485_R2.trim.paired.fastq.gz data/trimmed/SRR17909485_R2.trim.unpaired.fastq.gz \
    LEADING:22 TRAILING:22 SLIDINGWINDOW:6:22 MINLEN:32

Output

TrimmomaticPE: Started with arguments:
 -threads 2 data/SRR17909485_1.fastq.gz data/SRR17909485_2.fastq.gz data/trimmed/SRR17909485_R1.trim.paired.fastq.gz data/trimmed/SRR17909485_R1.trim.unpaired.fastq.gz data/trimmed/SRR17909485_R2.trim.paired.fastq.gz data/trimmed/SRR17909485_R2.trim.unpaired.fastq.gz LEADING:22 TRAILING:22 SLIDINGWINDOW:6:22 MINLEN:32
Quality encoding detected as phred33
Input Read Pairs: 1251776 Both Surviving: 1205173 (96.28%) Forward Only Surviving: 34920 (2.79%) Reverse Only Surviving: 2954 (0.24%) Dropped: 8729 (0.70%)
TrimmomaticPE: Completed successfully
  • LEADING:22: Removes low-quality bases from the start of the reads if they have a quality score lower than 22.
  • TRAILING:22: Removes low-quality bases from the end of the reads if they have a quality score lower than 22.
  • SLIDINGWINDOW:6:22: Performs a sliding window trimming. It scans the read with a 6-base wide window, cutting when the average quality per base drops below 22.
  • MINLEN:32: Discards reads that are shorter than 32 bases after trimming.

Input Read Pairs: 1251776
- Both Surviving: 1205173 (96.28%)
- Forward Only Surviving: 34920 (2.79%)
- Reverse Only Surviving: 2954 (0.24%)
- Dropped: 8729 (0.70%)

Step 4: Index reference

Input

bwa index data/reference/EcoliK12MG1655.fa

Step 5: Align to reference

Input

mkdir data/bam/

Input

bwa mem -t 2 -R '@RG\tID:1' \
    data/reference/EcoliK12MG1655.fa \
    data/trimmed/SRR17909485_R1.trim.paired.fastq.gz \
    data/trimmed/SRR17909485_R2.trim.paired.fastq.gz \
    | samtools view -b > data/bam/EcoliK12MG1655.SRR17909485.unsorted.bam

Step 6: Sort alignment

Input

samtools sort --threads 2 data/bam/EcoliK12MG1655.SRR17909485.unsorted.bam > \
    data/bam/EcoliK12MG1655.SRR17909485.sorted.bam

Step 7: Make bam index

Input

samtools index data/bam/EcoliK12MG1655.SRR17909485.sorted.bam

Step 8: Realign indels

Input

abra2 --threads 2 --mad 100 --mbq 24 \
    --ref data/reference/EcoliK12MG1655.fa \
    --in data/bam/EcoliK12MG1655.SRR17909485.sorted.bam \
    --out data/bam/EcoliK12MG1655.SRR17909485.final.bam

Step 9: Index final bam

Input

samtools index data/bam/EcoliK12MG1655.SRR17909485.final.bam

Step 10: Call variants

Input

mkdir data/vcf/

Input

bcftools mpileup -Ou --max-depth 5000 \
    -f data/reference/EcoliK12MG1655.fa \
    data/bam/EcoliK12MG1655.SRR17909485.final.bam \
    | bcftools call -mv --ploidy 1 -Ov \
    -o data/vcf/EcoliK12MG1655.SRR17909485.called.bcftools.vcf

Mpileup is actually a slightly different representation of alignment. Mpileup takes very simple things, it takes the number of reads aligned to each point and for each position in the reference genome it counts statistics - how many letters match the reference, how many mismatch, how many insertions, deletions.

Unfortunately bcftools is not multithreaded. If we do re-alignment with abra2, bcftools will work a bit faster.

bcftools --ploidy 1 - we tell the program that we have a haploid genome.

vcf = variant call format. This is actually a list of variations in a clear text format: chromosome, position, which letter was replaced by which letter, the quality of how much bcftools considers it important, some set of certain statistics (frequency of letter occurrence, etc.).

Input

tail data/vcf/EcoliK12MG1655.SRR17909485.called.bcftools.vcf

Output

U00096.3    3110421 .   C   A   225.417 .   DP=41;VDB=0.454064;SGB=-0.692914;MQSBZ=1.45774;MQ0F=0;AC=1;AN=1;DP4=0,0,8,17;MQ=59  GT:PL   1:255,0
U00096.3    3560455 .   C   CG  225.417 .   INDEL;IDV=82;IMF=1;DP=82;VDB=0.0327919;SGB=-0.693147;MQSBZ=0;BQBZ=0.885506;MQ0F=0;AC=1;AN=1;DP4=0,0,37,45;MQ=60 GT:PL   1:255,0
U00096.3    4093770 .   C   T   225.417 .   DP=141;VDB=0.961761;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,39,52;MQ=60  GT:PL   1:255,0
U00096.3    4161248 .   G   T   225.417 .   DP=117;VDB=0.400301;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,36,33;MQ=60  GT:PL   1:255,0
U00096.3    4164123 .   C   G   225.417 .   DP=130;VDB=0.322528;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,32,44;MQ=60  GT:PL   1:255,0
U00096.3    4296380 .   AC  ACGC    228.422 .   INDEL;IDV=98;IMF=0.989899;DP=99;VDB=8.47905e-05;SGB=-0.693147;RPBZ=-1.71529;MQBZ=1.83497;MQSBZ=-2.15139;BQBZ=-1.59515;SCBZ=-9.8995;MQ0F=0;AC=1;AN=1;DP4=0,1,47,51;MQ=54 GT:PL   1:255,0
U00096.3    4474834 .   A   G   225.417 .   DP=81;VDB=0.345282;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,21,26;MQ=60   GT:PL   1:255,0
U00096.3    4585480 .   G   A   225.417 .   DP=70;VDB=0.996826;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,20,26;MQ=60   GT:PL   1:255,0
U00096.3    4602509 .   C   T   225.417 .   DP=136;VDB=0.843259;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,42,41;MQ=60  GT:PL   1:255,0
U00096.3    4616669 .   G   T   225.417 .   DP=88;VDB=0.58582;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,25,31;MQ=60    GT:PL   1:255,0

Step 11: Make snpEff database

snpEff has a database of already well-sequenced, annotated genomes of many known organisms. snpEff loads the annotations and while annotating the vcf file, it checks which gene each particular variation falls into which position.
By E. coli snpEff has over 3500 databases, which is a lot.
The best way to handle this is to make your own database.

Input

mkdir -p data/EcoliK12MG1655/

Input

cp data/reference/EcoliK12MG1655.gb data/EcoliK12MG1655/genes.gbk

Input

echo "EcoliK12MG1655.genome : EcoliK12MG1655\nEcoliK12MG1655.chromosomes : EcoliK12MG1655.gb\nEcoliK12MG1655.codonTable : Standard" \
    > snpEff.config

Input

cat snpEff.config

Output

EcoliK12MG1655.genome : EcoliK12MG1655
EcoliK12MG1655.chromosomes : EcoliK12MG1655.gb
EcoliK12MG1655.codonTable : Standard
  1. Line 1 - under what name snpEff will know this reference genome (EcoliK12MG1655)
  2. Line 2 - the chromosomes from this genome (all contigs with all annotations) will be in the file EcoliK12MG1655.gb
  3. Line 3 - standard genetic code table

Input

snpEff build -c snpEff.config -genbank EcoliK12MG1655

Output

00:00:00 Codon table 'Standard' for genome 'EcoliK12MG1655'
    Protein check:  EcoliK12MG1655  OK: 4298    Not found: 17   Errors: 0   Error percentage: 0.0%

Step 12: Annotate variants

Input

snpEff ann -v EcoliK12MG1655  \
    data/vcf/EcoliK12MG1655.SRR17909485.called.bcftools.vcf > \
    data/vcf/EcoliK12MG1655.SRR17909485.annotated.vcf

Input

mv snpEff_genes.txt EcoliK12MG1655.SRR17909485.snpEff_genes.txt

Input

mv snpEff_summary.html EcoliK12MG1655.SRR17909485.snpEff_summary.html

Input

tail data/vcf/EcoliK12MG1655.SRR17909485.annotated.vcf

Output

U00096.3    3110421 .   C   A   225.417 .   DP=41;VDB=0.454064;SGB=-0.692914;MQSBZ=1.45774;MQ0F=0;AC=1;AN=1;DP4=0,0,8,17;MQ=59;ANN=A|upstream_gene_variant|MODIFIER|speC|b2965|transcript|b2965|protein_coding||c.-1266G>T|||||1266|,A|upstream_gene_variant|MODIFIER|yqgH|b4785|transcript|b4785|protein_coding||c.-1021G>T|||||1021|,A|upstream_gene_variant|MODIFIER|yqhJ|b4786|transcript|b4786|protein_coding||c.-845C>A|||||845|WARNING_TRANSCRIPT_NO_START_CODON,A|downstream_gene_variant|MODIFIER|mltC|b2963|transcript|b2963|protein_coding||c.*4909C>A|||||4909|,A|downstream_gene_variant|MODIFIER|nupG|b2964|transcript|b2964|protein_coding||c.*3451C>A|||||3451|,A|downstream_gene_variant|MODIFIER|yqgA|b2966|transcript|b2966|protein_coding||c.*161C>A|||||161|WARNING_TRANSCRIPT_NO_START_CODON,A|downstream_gene_variant|MODIFIER|yghD|b2968|transcript|b2968|protein_coding||c.*169G>T|||||169|,A|downstream_gene_variant|MODIFIER|yghE|b2969|transcript|b2969|protein_coding||c.*707G>T|||||707|WARNING_TRANSCRIPT_NO_START_CODON,A|downstream_gene_variant|MODIFIER|yghF|b2970|transcript|b2970|protein_coding||c.*1633G>T|||||1633|WARNING_TRANSCRIPT_NO_START_CODON,A|downstream_gene_variant|MODIFIER|yghG|b2971|transcript|b2971|protein_coding||c.*2646G>T|||||2646|,A|downstream_gene_variant|MODIFIER|pppA|b2972|transcript|b2972|protein_coding||c.*3122G>T|||||3122|,A|downstream_gene_variant|MODIFIER|yghJ|b4466|transcript|b4466|protein_coding||c.*4129G>T|||||4129|,A|intragenic_variant|MODIFIER|pheV|b2967|gene_variant|b2967|||n.3110421C>A||||||   GT:PL   1:255,0
U00096.3    3560455 .   C   CG  225.417 .   INDEL;IDV=82;IMF=1;DP=82;VDB=0.0327919;SGB=-0.693147;MQSBZ=0;BQBZ=0.885506;MQ0F=0;AC=1;AN=1;DP4=0,0,37,45;MQ=60;ANN=CG|upstream_gene_variant|MODIFIER|rtcA|b4475|transcript|b4475|protein_coding||c.-3607_-3606insC|||||3607|,CG|upstream_gene_variant|MODIFIER|rtcB|b3421|transcript|b3421|protein_coding||c.-2377_-2376insC|||||2377|,CG|upstream_gene_variant|MODIFIER|glpD|b3426|transcript|b3426|protein_coding||c.-1558_-1557insG|||||1557|,CG|downstream_gene_variant|MODIFIER|malT|b3418|transcript|b3418|protein_coding||c.*4665_*4666insG|||||4666|,CG|downstream_gene_variant|MODIFIER|rtcR|b3422|transcript|b3422|protein_coding||c.*589_*590insG|||||590|,CG|downstream_gene_variant|MODIFIER|glpG|b3424|transcript|b3424|protein_coding||c.*166_*167insC|||||166|,CG|downstream_gene_variant|MODIFIER|glpE|b3425|transcript|b3425|protein_coding||c.*1041_*1042insC|||||1041|,CG|downstream_gene_variant|MODIFIER|yzgL|b3427|transcript|b3427|protein_coding||c.*3268_*3269insC|||||3268|,CG|downstream_gene_variant|MODIFIER|glgP|b3428|transcript|b3428|protein_coding||c.*3678_*3679insC|||||3678|,CG|intragenic_variant|MODIFIER|glpR|b3423|gene_variant|b3423|||n.3560455_3560456insG||||||  GT:PL   1:255,0
U00096.3    4093770 .   C   T   225.417 .   DP=141;VDB=0.961761;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,39,52;MQ=60;ANN=T|missense_variant|MODERATE|rhaD|b3902|transcript|b3902|protein_coding|1/1|c.503G>A|p.Gly168Asp|503/825|503/825|168/274||,T|upstream_gene_variant|MODIFIER|frvR|b3897|transcript|b3897|protein_coding||c.-3915G>A|||||3915|,T|upstream_gene_variant|MODIFIER|frvX|b3898|transcript|b3898|protein_coding||c.-2845G>A|||||2845|,T|upstream_gene_variant|MODIFIER|frvB|b3899|transcript|b3899|protein_coding||c.-1404G>A|||||1404|,T|upstream_gene_variant|MODIFIER|frvA|b3900|transcript|b3900|protein_coding||c.-947G>A|||||947|,T|upstream_gene_variant|MODIFIER|rhaM|b3901|transcript|b3901|protein_coding||c.-332G>A|||||332|,T|upstream_gene_variant|MODIFIER|rhaS|b3905|transcript|b3905|protein_coding||c.-3966C>T|||||3966|,T|upstream_gene_variant|MODIFIER|rhaR|b3906|transcript|b3906|protein_coding||c.-4876C>T|||||4876|WARNING_TRANSCRIPT_NO_START_CODON,T|downstream_gene_variant|MODIFIER|rhaA|b3903|transcript|b3903|protein_coding||c.*953G>A|||||953|,T|downstream_gene_variant|MODIFIER|rhaB|b3904|transcript|b3904|protein_coding||c.*2209G>A|||||2209|   GT:PL   1:255,0
U00096.3    4161248 .   G   T   225.417 .   DP=117;VDB=0.400301;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,36,33;MQ=60;ANN=T|missense_variant|MODERATE|fabR|b3963|transcript|b3963|protein_coding|1/1|c.182G>T|p.Gly61Val|182/705|182/705|61/234||,T|upstream_gene_variant|MODIFIER|sthA|b3962|transcript|b3962|protein_coding||c.-458C>A|||||458|,T|upstream_gene_variant|MODIFIER|yijD|b3964|transcript|b3964|protein_coding||c.-523G>T|||||523|,T|upstream_gene_variant|MODIFIER|btuB|b3966|transcript|b3966|protein_coding||c.-2391G>T|||||2391|,T|upstream_gene_variant|MODIFIER|murI|b3967|transcript|b3967|protein_coding||c.-4180G>T|||||4180|,T|downstream_gene_variant|MODIFIER|argB|b3959|transcript|b3959|protein_coding||c.*4459G>T|||||4459|,T|downstream_gene_variant|MODIFIER|argH|b3960|transcript|b3960|protein_coding||c.*3025G>T|||||3025|,T|downstream_gene_variant|MODIFIER|oxyR|b3961|transcript|b3961|protein_coding||c.*1841G>T|||||1841|,T|downstream_gene_variant|MODIFIER|trmA|b3965|transcript|b3965|protein_coding||c.*922C>A|||||922|    GT:PL   1:255,0
U00096.3    4164123 .   C   G   225.417 .   DP=130;VDB=0.322528;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,32,44;MQ=60;ANN=G|missense_variant|MODERATE|btuB|b3966|transcript|b3966|protein_coding|1/1|c.485C>G|p.Ala162Gly|485/1845|485/1845|162/614||,G|upstream_gene_variant|MODIFIER|sthA|b3962|transcript|b3962|protein_coding||c.-3333G>C|||||3333|,G|upstream_gene_variant|MODIFIER|trmA|b3965|transcript|b3965|protein_coding||c.-853G>C|||||853|,G|upstream_gene_variant|MODIFIER|murI|b3967|transcript|b3967|protein_coding||c.-1305C>G|||||1305|,G|downstream_gene_variant|MODIFIER|oxyR|b3961|transcript|b3961|protein_coding||c.*4716C>G|||||4716|,G|downstream_gene_variant|MODIFIER|fabR|b3963|transcript|b3963|protein_coding||c.*2352C>G|||||2352|,G|downstream_gene_variant|MODIFIER|yijD|b3964|transcript|b3964|protein_coding||c.*1993C>G|||||1993|  GT:PL   1:255,0
U00096.3    4296380 .   AC  ACGC    228.422 .   INDEL;IDV=98;IMF=0.989899;DP=99;VDB=8.47905e-05;SGB=-0.693147;RPBZ=-1.71529;MQBZ=1.83497;MQSBZ=-2.15139;BQBZ=-1.59515;SCBZ=-9.8995;MQ0F=0;AC=1;AN=1;DP4=0,1,47,51;MQ=54;ANN=ACGC|downstream_gene_variant|MODIFIER|nrfD|b4073|transcript|b4073|protein_coding||c.*4949_*4950insGC|||||4950|,ACGC|downstream_gene_variant|MODIFIER|nrfE|b4074|transcript|b4074|protein_coding||c.*3211_*3212insGC|||||3212|WARNING_TRANSCRIPT_NO_START_CODON,ACGC|downstream_gene_variant|MODIFIER|nrfF|b4075|transcript|b4075|protein_coding||c.*2835_*2836insGC|||||2836|,ACGC|downstream_gene_variant|MODIFIER|nrfG|b4076|transcript|b4076|protein_coding||c.*2242_*2243insGC|||||2243|,ACGC|downstream_gene_variant|MODIFIER|gltP|b4077|transcript|b4077|protein_coding||c.*587_*588insGC|||||588|,ACGC|downstream_gene_variant|MODIFIER|yjcO|b4078|transcript|b4078|protein_coding||c.*54_*55insGC|||||54|,ACGC|downstream_gene_variant|MODIFIER|fdhF|b4079|transcript|b4079|protein_coding||c.*837_*838insGC|||||837|,ACGC|downstream_gene_variant|MODIFIER|mdtP|b4080|transcript|b4080|protein_coding||c.*3182_*3183insGC|||||3182|,ACGC|downstream_gene_variant|MODIFIER|mdtO|b4081|transcript|b4081|protein_coding||c.*4645_*4646insGC|||||4645|,ACGC|intergenic_region|MODIFIER|gltP-yjcO|b4077-b4078|intergenic_region|b4077-b4078|||n.4296381_4296382insGC||||||  GT:PL   1:255,0
U00096.3    4474834 .   A   G   225.417 .   DP=81;VDB=0.345282;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,21,26;MQ=60;ANN=G|upstream_gene_variant|MODIFIER|ridA|b4243|transcript|b4243|protein_coding||c.-3921T>C|||||3921|,G|upstream_gene_variant|MODIFIER|pyrI|b4244|transcript|b4244|protein_coding||c.-3387T>C|||||3387|,G|upstream_gene_variant|MODIFIER|pyrB|b4245|transcript|b4245|protein_coding||c.-2439T>C|||||2439|,G|upstream_gene_variant|MODIFIER|pyrL|b4246|transcript|b4246|protein_coding||c.-2301T>C|||||2301|,G|upstream_gene_variant|MODIFIER|yjgH|b4248|transcript|b4248|protein_coding||c.-1625T>C|||||1625|,G|upstream_gene_variant|MODIFIER|bdcA|b4249|transcript|b4249|protein_coding||c.-781T>C|||||781|,G|upstream_gene_variant|MODIFIER|tabA|b4252|transcript|b4252|protein_coding||c.-28A>G|||||28|,G|upstream_gene_variant|MODIFIER|yjgL|b4253|transcript|b4253|protein_coding||c.-603A>G|||||603|,G|upstream_gene_variant|MODIFIER|rraB|b4255|transcript|b4255|protein_coding||c.-3639A>G|||||3639|,G|upstream_gene_variant|MODIFIER|yjgN|b4257|transcript|b4257|protein_coding||c.-4896A>G|||||4896|,G|downstream_gene_variant|MODIFIER|mgtA|b4242|transcript|b4242|protein_coding||c.*4513A>G|||||4513|,G|downstream_gene_variant|MODIFIER|bdcR|b4251|transcript|b4251|protein_coding||c.*117A>G|||||117|,G|downstream_gene_variant|MODIFIER|argI|b4254|transcript|b4254|protein_coding||c.*2473T>C|||||2473|,G|downstream_gene_variant|MODIFIER|yjgM|b4256|transcript|b4256|protein_coding||c.*4200T>C|||||4200|,G|intergenic_region|MODIFIER|bdcR-tabA|b4251-b4252|intergenic_region|b4251-b4252|||n.4474834A>G||||||   GT:PL   1:255,0
U00096.3    4585480 .   G   A   225.417 .   DP=70;VDB=0.996826;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,20,26;MQ=60;ANN=A|stop_gained|HIGH|hsdR|b4350|transcript|b4350|protein_coding|1/1|c.1282C>T|p.Gln428*|1282/3513|1282/3513|428/1170||,A|upstream_gene_variant|MODIFIER|hsdS|b4348|transcript|b4348|protein_coding||c.-4018C>T|||||4018|,A|upstream_gene_variant|MODIFIER|hsdM|b4349|transcript|b4349|protein_coding||c.-2432C>T|||||2432|,A|upstream_gene_variant|MODIFIER|mrr|b4351|transcript|b4351|protein_coding||c.-1469G>A|||||1469|,A|downstream_gene_variant|MODIFIER|yjiA|b4352|transcript|b4352|protein_coding||c.*2429C>T|||||2429|,A|downstream_gene_variant|MODIFIER|yjiX|b4353|transcript|b4353|protein_coding||c.*3396C>T|||||3396|,A|downstream_gene_variant|MODIFIER|btsT|b4354|transcript|b4354|protein_coding||c.*3649C>T|||||3649| GT:PL   1:255,0
U00096.3    4602509 .   C   T   225.417 .   DP=136;VDB=0.843259;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,42,41;MQ=60;ANN=T|stop_gained|HIGH|yjjP|b4364|transcript|b4364|protein_coding|1/1|c.350G>A|p.Trp117*|350/771|350/771|117/256||,T|upstream_gene_variant|MODIFIER|opgB|b4359|transcript|b4359|protein_coding||c.-3068G>A|||||3068|WARNING_TRANSCRIPT_NO_START_CODON,T|upstream_gene_variant|MODIFIER|yjjA|b4360|transcript|b4360|protein_coding||c.-2320G>A|||||2320|,T|upstream_gene_variant|MODIFIER|dnaC|b4361|transcript|b4361|protein_coding||c.-1534G>A|||||1534|,T|upstream_gene_variant|MODIFIER|dnaT|b4362|transcript|b4362|protein_coding||c.-992G>A|||||992|,T|upstream_gene_variant|MODIFIER|yjjB|b4363|transcript|b4363|protein_coding||c.-412G>A|||||412|,T|upstream_gene_variant|MODIFIER|yjjQ|b4365|transcript|b4365|protein_coding||c.-968C>T|||||968|,T|upstream_gene_variant|MODIFIER|bglJ|b4366|transcript|b4366|protein_coding||c.-1651C>T|||||1651|,T|upstream_gene_variant|MODIFIER|yjjZ|b4567|transcript|b4567|protein_coding||c.-3295C>T|||||3295|,T|downstream_gene_variant|MODIFIER|fhuF|b4367|transcript|b4367|protein_coding||c.*2366G>A|||||2366|,T|downstream_gene_variant|MODIFIER|rsmC|b4371|transcript|b4371|protein_coding||c.*4160G>A|||||4160|    GT:PL   1:255,0
U00096.3    4616669 .   G   T   225.417 .   DP=88;VDB=0.58582;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,25,31;MQ=60;ANN=T|missense_variant|MODERATE|yjjI|b4380|transcript|b4380|protein_coding|1/1|c.397C>A|p.Leu133Ile|397/1551|397/1551|133/516||,T|upstream_gene_variant|MODIFIER|yjjW|b4379|transcript|b4379|protein_coding||c.-1126C>A|||||1126|,T|upstream_gene_variant|MODIFIER|deoC|b4381|transcript|b4381|protein_coding||c.-654G>T|||||654|,T|upstream_gene_variant|MODIFIER|deoA|b4382|transcript|b4382|protein_coding||c.-1560G>T|||||1560|WARNING_TRANSCRIPT_NO_START_CODON,T|upstream_gene_variant|MODIFIER|deoB|b4383|transcript|b4383|protein_coding||c.-2934G>T|||||2934|,T|upstream_gene_variant|MODIFIER|deoD|b4384|transcript|b4384|protein_coding||c.-4214G>T|||||4214|,T|downstream_gene_variant|MODIFIER|osmY|b4376|transcript|b4376|protein_coding||c.*4668G>T|||||4668|,T|downstream_gene_variant|MODIFIER|ytjA|b4568|transcript|b4568|protein_coding||c.*4380G>T|||||4380|,T|downstream_gene_variant|MODIFIER|yjjU|b4377|transcript|b4377|protein_coding||c.*3185G>T|||||3185|WARNING_TRANSCRIPT_NO_START_CODON,T|downstream_gene_variant|MODIFIER|yjjV|b4378|transcript|b4378|protein_coding||c.*2409G>T|||||2409|WARNING_TRANSCRIPT_NO_START_CODON GT:PL   1:255,0

The vcf file became significantly larger because the ANN (annotation) line appeared after all the technical information
Example:

ANN=T|missense_variant|MODERATE|yjjI|b4380|transcript|b4380|protein_coding|1/1|c.397C>A|p.Leu133Ile.
  • There's been a substitution of a letter for the T.
  • This is a missence variant mutation.
  • Its effect is MODERATE.
  • yjjI - gene name
  • b4380 - number of the transcript in which the substitution occurred.
  • protein_coding - this is the protein coding site
  • c.397C>A - replacement in nucleotide sequence
  • p.Leu133Ile - subsequent substitution in protein sequence (133 leucine is replaced by isoleucine)

Step 13: Filter high and moderate effect mutations

Let's filter out high and moderate effect mutations:
- nonsense
- missense
- frameshifts

Input

SnpSift filter "(ANN[*].IMPACT has 'HIGH') | (ANN[*].IMPACT has 'MODERATE')" data/vcf/EcoliK12MG1655.SRR17909485.annotated.vcf > data/vcf/EcoliK12MG1655.SRR17909485.higheffect.vcf

Input

tail data/vcf/EcoliK12MG1655.SRR17909485.higheffect.vcf

Output

U00096.3    705013  .   T   C   225.417 .   DP=103;VDB=0.0387749;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,30,36;MQ=60;ANN=C|missense_variant|MODERATE|nagE|b0679|transcript|b0679|protein_coding|1/1|c.1070T>C|p.Leu357Ser|1070/1947|1070/1947|357/648||,C|upstream_gene_variant|MODIFIER|umpH|b0675|transcript|b0675|protein_coding||c.-4687A>G|||||4687|,C|upstream_gene_variant|MODIFIER|nagC|b0676|transcript|b0676|protein_coding||c.-3419A>G|||||3419|,C|upstream_gene_variant|MODIFIER|nagA|b0677|transcript|b0677|protein_coding||c.-2262A>G|||||2262|,C|upstream_gene_variant|MODIFIER|nagB|b0678|transcript|b0678|protein_coding||c.-1402A>G|||||1402|,C|upstream_gene_variant|MODIFIER|glnS|b0680|transcript|b0680|protein_coding||c.-1080T>C|||||1080|,C|upstream_gene_variant|MODIFIER|chiP|b0681|transcript|b0681|protein_coding||c.-3321T>C|||||3321|,C|upstream_gene_variant|MODIFIER|chiQ|b0682|transcript|b0682|protein_coding||c.-4777T>C|||||4777|    GT:PL   1:255,0
U00096.3    1337394 .   A   G   225.417 .   DP=65;VDB=0.915279;SGB=-0.693146;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,24,19;MQ=60;ANN=G|missense_variant|MODERATE|acnA|b1276|transcript|b1276|protein_coding|1/1|c.1564A>G|p.Ser522Gly|1564/2676|1564/2676|522/891||,G|upstream_gene_variant|MODIFIER|pgpB|b1278|transcript|b1278|protein_coding||c.-1936A>G|||||1936|,G|upstream_gene_variant|MODIFIER|lapA|b1279|transcript|b1279|protein_coding||c.-2849A>G|||||2849|WARNING_TRANSCRIPT_NO_START_CODON,G|upstream_gene_variant|MODIFIER|lapB|b1280|transcript|b1280|protein_coding||c.-3164A>G|||||3164|,G|upstream_gene_variant|MODIFIER|pyrF|b1281|transcript|b1281|protein_coding||c.-4527A>G|||||4527|,G|downstream_gene_variant|MODIFIER|topA|b1274|transcript|b1274|protein_coding||c.*3749A>G|||||3749|,G|downstream_gene_variant|MODIFIER|cysB|b1275|transcript|b1275|protein_coding||c.*2565A>G|||||2565|,G|downstream_gene_variant|MODIFIER|ymiA|b4522|transcript|b4522|protein_coding||c.*2106A>G|||||2106|,G|downstream_gene_variant|MODIFIER|yciX|b4523|transcript|b4523|protein_coding||c.*1936A>G|||||1936|,G|downstream_gene_variant|MODIFIER|ymiC|b4741|transcript|b4741|protein_coding||c.*1727A>G|||||1727|,G|downstream_gene_variant|MODIFIER|ribA|b1277|transcript|b1277|protein_coding||c.*1176T>C|||||1176|   GT:PL   1:255,0
U00096.3    1514951 .   G   T   225.417 .   DP=68;VDB=0.981287;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,18,30;MQ=60;ANN=T|missense_variant|MODERATE|ydcV|b1443|transcript|b1443|protein_coding|1/1|c.190G>T|p.Ala64Ser|190/795|190/795|64/264||,T|upstream_gene_variant|MODIFIER|patD|b1444|transcript|b1444|protein_coding||c.-627G>T|||||627|,T|upstream_gene_variant|MODIFIER|ortT|b1445|transcript|b1445|protein_coding||c.-2438G>T|||||2438|,T|upstream_gene_variant|MODIFIER|ydcY|b1446|transcript|b1446|protein_coding||c.-2697G>T|||||2697|,T|upstream_gene_variant|MODIFIER|curA|b1449|transcript|b1449|protein_coding||c.-4076G>T|||||4076|,T|downstream_gene_variant|MODIFIER|ydcR|b1439|transcript|b1439|protein_coding||c.*3542G>T|||||3542|,T|downstream_gene_variant|MODIFIER|ydcS|b1440|transcript|b1440|protein_coding||c.*2152G>T|||||2152|,T|downstream_gene_variant|MODIFIER|ydcT|b1441|transcript|b1441|protein_coding||c.*1121G>T|||||1121|,T|downstream_gene_variant|MODIFIER|ydcU|b1442|transcript|b1442|protein_coding||c.*179G>T|||||179|,T|downstream_gene_variant|MODIFIER|yncL|b4598|transcript|b4598|protein_coding||c.*2148C>A|||||2148|,T|downstream_gene_variant|MODIFIER|ydcZ|b1447|transcript|b1447|protein_coding||c.*2931C>A|||||2931|,T|downstream_gene_variant|MODIFIER|mnaT|b1448|transcript|b1448|protein_coding||c.*3377C>A|||||3377|   GT:PL   1:255,0
U00096.3    2405959 .   G   T   225.417 .   DP=116;VDB=0.00680486;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,42,33;MQ=60;ANN=T|missense_variant|MODERATE|lrhA|b2289|transcript|b2289|protein_coding|1/1|c.683C>A|p.Ala228Glu|683/939|683/939|228/312||,T|upstream_gene_variant|MODIFIER|nuoF|b2284|transcript|b2284|protein_coding||c.-4404C>A|||||4404|,T|upstream_gene_variant|MODIFIER|nuoE|b2285|transcript|b2285|protein_coding||c.-3907C>A|||||3907|,T|upstream_gene_variant|MODIFIER|nuoC|b2286|transcript|b2286|protein_coding||c.-2114C>A|||||2114|,T|upstream_gene_variant|MODIFIER|nuoB|b2287|transcript|b2287|protein_coding||c.-1346C>A|||||1346|,T|upstream_gene_variant|MODIFIER|nuoA|b2288|transcript|b2288|protein_coding||c.-887C>A|||||887|,T|upstream_gene_variant|MODIFIER|alaA|b2290|transcript|b2290|protein_coding||c.-1602G>T|||||1602|,T|upstream_gene_variant|MODIFIER|yfbR|b2291|transcript|b2291|protein_coding||c.-2903G>T|||||2903|,T|downstream_gene_variant|MODIFIER|yfbS|b2292|transcript|b2292|protein_coding||c.*3561C>A|||||3561|WARNING_TRANSCRIPT_NO_START_CODON GT:PL   1:255,0
U00096.3    4093770 .   C   T   225.417 .   DP=141;VDB=0.961761;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,39,52;MQ=60;ANN=T|missense_variant|MODERATE|rhaD|b3902|transcript|b3902|protein_coding|1/1|c.503G>A|p.Gly168Asp|503/825|503/825|168/274||,T|upstream_gene_variant|MODIFIER|frvR|b3897|transcript|b3897|protein_coding||c.-3915G>A|||||3915|,T|upstream_gene_variant|MODIFIER|frvX|b3898|transcript|b3898|protein_coding||c.-2845G>A|||||2845|,T|upstream_gene_variant|MODIFIER|frvB|b3899|transcript|b3899|protein_coding||c.-1404G>A|||||1404|,T|upstream_gene_variant|MODIFIER|frvA|b3900|transcript|b3900|protein_coding||c.-947G>A|||||947|,T|upstream_gene_variant|MODIFIER|rhaM|b3901|transcript|b3901|protein_coding||c.-332G>A|||||332|,T|upstream_gene_variant|MODIFIER|rhaS|b3905|transcript|b3905|protein_coding||c.-3966C>T|||||3966|,T|upstream_gene_variant|MODIFIER|rhaR|b3906|transcript|b3906|protein_coding||c.-4876C>T|||||4876|WARNING_TRANSCRIPT_NO_START_CODON,T|downstream_gene_variant|MODIFIER|rhaA|b3903|transcript|b3903|protein_coding||c.*953G>A|||||953|,T|downstream_gene_variant|MODIFIER|rhaB|b3904|transcript|b3904|protein_coding||c.*2209G>A|||||2209|   GT:PL   1:255,0
U00096.3    4161248 .   G   T   225.417 .   DP=117;VDB=0.400301;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,36,33;MQ=60;ANN=T|missense_variant|MODERATE|fabR|b3963|transcript|b3963|protein_coding|1/1|c.182G>T|p.Gly61Val|182/705|182/705|61/234||,T|upstream_gene_variant|MODIFIER|sthA|b3962|transcript|b3962|protein_coding||c.-458C>A|||||458|,T|upstream_gene_variant|MODIFIER|yijD|b3964|transcript|b3964|protein_coding||c.-523G>T|||||523|,T|upstream_gene_variant|MODIFIER|btuB|b3966|transcript|b3966|protein_coding||c.-2391G>T|||||2391|,T|upstream_gene_variant|MODIFIER|murI|b3967|transcript|b3967|protein_coding||c.-4180G>T|||||4180|,T|downstream_gene_variant|MODIFIER|argB|b3959|transcript|b3959|protein_coding||c.*4459G>T|||||4459|,T|downstream_gene_variant|MODIFIER|argH|b3960|transcript|b3960|protein_coding||c.*3025G>T|||||3025|,T|downstream_gene_variant|MODIFIER|oxyR|b3961|transcript|b3961|protein_coding||c.*1841G>T|||||1841|,T|downstream_gene_variant|MODIFIER|trmA|b3965|transcript|b3965|protein_coding||c.*922C>A|||||922|    GT:PL   1:255,0
U00096.3    4164123 .   C   G   225.417 .   DP=130;VDB=0.322528;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,32,44;MQ=60;ANN=G|missense_variant|MODERATE|btuB|b3966|transcript|b3966|protein_coding|1/1|c.485C>G|p.Ala162Gly|485/1845|485/1845|162/614||,G|upstream_gene_variant|MODIFIER|sthA|b3962|transcript|b3962|protein_coding||c.-3333G>C|||||3333|,G|upstream_gene_variant|MODIFIER|trmA|b3965|transcript|b3965|protein_coding||c.-853G>C|||||853|,G|upstream_gene_variant|MODIFIER|murI|b3967|transcript|b3967|protein_coding||c.-1305C>G|||||1305|,G|downstream_gene_variant|MODIFIER|oxyR|b3961|transcript|b3961|protein_coding||c.*4716C>G|||||4716|,G|downstream_gene_variant|MODIFIER|fabR|b3963|transcript|b3963|protein_coding||c.*2352C>G|||||2352|,G|downstream_gene_variant|MODIFIER|yijD|b3964|transcript|b3964|protein_coding||c.*1993C>G|||||1993|  GT:PL   1:255,0
U00096.3    4585480 .   G   A   225.417 .   DP=70;VDB=0.996826;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,20,26;MQ=60;ANN=A|stop_gained|HIGH|hsdR|b4350|transcript|b4350|protein_coding|1/1|c.1282C>T|p.Gln428*|1282/3513|1282/3513|428/1170||,A|upstream_gene_variant|MODIFIER|hsdS|b4348|transcript|b4348|protein_coding||c.-4018C>T|||||4018|,A|upstream_gene_variant|MODIFIER|hsdM|b4349|transcript|b4349|protein_coding||c.-2432C>T|||||2432|,A|upstream_gene_variant|MODIFIER|mrr|b4351|transcript|b4351|protein_coding||c.-1469G>A|||||1469|,A|downstream_gene_variant|MODIFIER|yjiA|b4352|transcript|b4352|protein_coding||c.*2429C>T|||||2429|,A|downstream_gene_variant|MODIFIER|yjiX|b4353|transcript|b4353|protein_coding||c.*3396C>T|||||3396|,A|downstream_gene_variant|MODIFIER|btsT|b4354|transcript|b4354|protein_coding||c.*3649C>T|||||3649| GT:PL   1:255,0
U00096.3    4602509 .   C   T   225.417 .   DP=136;VDB=0.843259;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,42,41;MQ=60;ANN=T|stop_gained|HIGH|yjjP|b4364|transcript|b4364|protein_coding|1/1|c.350G>A|p.Trp117*|350/771|350/771|117/256||,T|upstream_gene_variant|MODIFIER|opgB|b4359|transcript|b4359|protein_coding||c.-3068G>A|||||3068|WARNING_TRANSCRIPT_NO_START_CODON,T|upstream_gene_variant|MODIFIER|yjjA|b4360|transcript|b4360|protein_coding||c.-2320G>A|||||2320|,T|upstream_gene_variant|MODIFIER|dnaC|b4361|transcript|b4361|protein_coding||c.-1534G>A|||||1534|,T|upstream_gene_variant|MODIFIER|dnaT|b4362|transcript|b4362|protein_coding||c.-992G>A|||||992|,T|upstream_gene_variant|MODIFIER|yjjB|b4363|transcript|b4363|protein_coding||c.-412G>A|||||412|,T|upstream_gene_variant|MODIFIER|yjjQ|b4365|transcript|b4365|protein_coding||c.-968C>T|||||968|,T|upstream_gene_variant|MODIFIER|bglJ|b4366|transcript|b4366|protein_coding||c.-1651C>T|||||1651|,T|upstream_gene_variant|MODIFIER|yjjZ|b4567|transcript|b4567|protein_coding||c.-3295C>T|||||3295|,T|downstream_gene_variant|MODIFIER|fhuF|b4367|transcript|b4367|protein_coding||c.*2366G>A|||||2366|,T|downstream_gene_variant|MODIFIER|rsmC|b4371|transcript|b4371|protein_coding||c.*4160G>A|||||4160|    GT:PL   1:255,0
U00096.3    4616669 .   G   T   225.417 .   DP=88;VDB=0.58582;SGB=-0.693147;MQSBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,25,31;MQ=60;ANN=T|missense_variant|MODERATE|yjjI|b4380|transcript|b4380|protein_coding|1/1|c.397C>A|p.Leu133Ile|397/1551|397/1551|133/516||,T|upstream_gene_variant|MODIFIER|yjjW|b4379|transcript|b4379|protein_coding||c.-1126C>A|||||1126|,T|upstream_gene_variant|MODIFIER|deoC|b4381|transcript|b4381|protein_coding||c.-654G>T|||||654|,T|upstream_gene_variant|MODIFIER|deoA|b4382|transcript|b4382|protein_coding||c.-1560G>T|||||1560|WARNING_TRANSCRIPT_NO_START_CODON,T|upstream_gene_variant|MODIFIER|deoB|b4383|transcript|b4383|protein_coding||c.-2934G>T|||||2934|,T|upstream_gene_variant|MODIFIER|deoD|b4384|transcript|b4384|protein_coding||c.-4214G>T|||||4214|,T|downstream_gene_variant|MODIFIER|osmY|b4376|transcript|b4376|protein_coding||c.*4668G>T|||||4668|,T|downstream_gene_variant|MODIFIER|ytjA|b4568|transcript|b4568|protein_coding||c.*4380G>T|||||4380|,T|downstream_gene_variant|MODIFIER|yjjU|b4377|transcript|b4377|protein_coding||c.*3185G>T|||||3185|WARNING_TRANSCRIPT_NO_START_CODON,T|downstream_gene_variant|MODIFIER|yjjV|b4378|transcript|b4378|protein_coding||c.*2409G>T|||||2409|WARNING_TRANSCRIPT_NO_START_CODON GT:PL   1:255,0