QuickVariants

Code for paper "Fast and Accurate Variant Identification Tool for Sequencing-Based Studies"

QuickVariants is a fast and accurate variant identification tool, designed to summarize allele information from read alignments without discarding or filtering the data.

Install

Requirement: Java \ Please download the latest QuickVariants here

You may install java by conda install conda-forge::openjdk\ You may install java and QuickVariants by conda install caozhichongchong::quick-variants\ QuickVariants can be found at $Conda_env_location/bin/quick-variants-VERSION.jar

Usage

java -Xms10g -Xmx10g -jar quick-variants-VERSION.jar [--out-vcf <out.vcf>] [--out-mutations <out.txt>] --reference <ref.fasta> --in-sam <input.sam> --num-threads num_threads [options]

This command converts a SAM file to other formats, most notably .vcf.

Input

• --reference <file>: The reference to use. Should be in .fasta/.fa/.fna or .fastq/.fq format or a .gz of one of those formats.

Output formats\ Summary by reference position, mutation, genome, and raw output are possible.

• Options for output files, mutation counts, exclusion of non-mutations, and more.

Summary by reference position

• --out-vcf <file> output file to generate containing a description of mutation counts by position
• --vcf-exclude-non-mutations if set, the output vcf file will exclude positions where no mutations were detected
• --distinguish-query-ends <fraction> (default 0.1) In the output vcf file, we separately display which queries aligned at each position with of the end of the query and which didn't.

Summary by mutation

• --out-mutations <file> output file to generate listing the mutations of the queries compared to the reference genome

• --distinguish-query-ends <fraction> (default 0.1) When detecting indels, only consider the middle of each query

• --snp-threshold <min total depth> <min supporting depth fraction> (default 5, 0.9) The minimum total depth and minimum supporting depth fraction required at a position to report it as a point mutation

• --indel-start-threshold <min total depth> <min supporting depth fraction> (default 1, 0.8) The minimum total (middle) depth and minimum supporting depth fraction required at a position to report it as the start of an insertion or deletion

• --indel-continue-threshold <min total depth> <min supporting depth fraction> (default 1, 0.7) The minimum total (middle) depth and minimum supporting depth fraction required at a position to report it as a continuation of an insertion or deletion

• --indel-threshold <min total depth> <min supporting depth fraction> Alias for --indel-start-threshold and --indel-continue-threshold

Summary by genome

• --out-refs-map-count <file> the output file to summarize the number of reads mapped to each combination of references

Raw output

• --out-sam <file> the output file in SAM format

• --out-unaligned <file> output file containing unaligned reads. Must have a .fasta or .fastq extension

• --no-output if no output is requested, skip writing output rather than throwing an error

Debugging

• -v, --verbose output diagnostic information

• -vv more diagnostic information

• --verbosity-auto set verbosity flags dynamically and automatically based on the data and alignment

Multiple output formats may be specified during a single run; for example:

• --out-sam out.sam --out-unaligned out.fastq

Others

• --num-threads <count> number of threads to use at once for processing. Higher values will run more quickly on a system that has that many CPUs available.

Test

java -jar quick-variants-1.1.0.jar --out-vcf Fig4Example1.vcf --reference examples/Fig4/reference.fasta --in-sam examples/Fig4/Example1/90.sam
java -jar quick-variants-1.1.0.jar --out-vcf Fig4Example2.vcf --reference examples/Fig4/reference.fasta --in-sam examples/Fig4/Example2/86.sam
java -jar quick-variants-1.1.0.jar --out-vcf Fig5Example1.vcf --reference examples/Fig5/reference.fasta --in-sam examples/Fig5/Example1/99.sam
java -jar quick-variants-1.1.0.jar --out-vcf Fig5Example2.vcf --reference examples/Fig5/reference.fasta --in-sam examples/Fig5/Example2/102.sam
java -jar quick-variants-1.1.0.jar --out-vcf Fig5Example3.vcf --reference examples/Fig5/reference.fasta --in-sam examples/Fig5/Example3/9.sam
java -jar quick-variants-1.1.0.jar --out-vcf Fig5Example4.vcf --reference examples/Fig5/reference.fasta --in-sam examples/Fig5/Example4/47.sam
java -jar quick-variants-1.1.0.jar --out-vcf Fig6Example1.vcf --reference examples/Fig6/reference.fasta --in-sam examples/Fig6/Example1/88.sam
java -jar quick-variants-1.1.0.jar --out-vcf Fig6Example2.vcf --reference examples/Fig6/reference.fasta --in-sam examples/Fig6/Example2/90.sam
java -jar quick-variants-1.1.0.jar --out-vcf Fig6Example3.vcf --reference examples/Fig6/reference.fasta --in-sam examples/Fig6/Example3/9.sam

• You can compare your results to pre-generated VCF files located in example/test_results/.

  filtering point mutations and indels

  python QuickVariants_Pointmutationfilter.py -i Your_QuickVariants_Output_Folder
  python QuickVariants_Indelfilter.py -i Your_QuickVariants_Output_Folder

• af, allele-frequency minimum allele frequency
• rd, read-depth minimum read depth
• Testing

  unzip test_vcf.zip
  python QuickVariants_Pointmutationfilter.py
  python QuickVariants_Indelfilter.py

Additional scripts and models

The benchmark_scripts folder contains code used to construct the benchmark dataset, filter SNPs and indels in VCF files, and analyze VCF files.

• SNP_model.py: Insert in silico mutations and indels randomly into reference genomes, and generate alignment code.
• SNPfilter.py: Filter SNPs in VCF files generated by QuickVariants and bcftools.
• Indelfiter.py: Filter indels in VCF files generated by QuickVariants and bcftools, and summarize the number of false positives and false negatives for each dataset.
• SNP_model_compare.py: Summarize the number of false positive and false negative SNPs for each dataset.
• VCFAnalysis.ipynb: Code to analyze point mutations and indels detected from benchmark datasets.
• COVID_MG.ipynb: Code to analyze point mutations and indels detected from SARS-COV-2 sewage MG real data.

Analyzing benchmark datasets used in this study\ Please download benchmark datasets here\ Requirements: bowtie2, bwa, minimap2, samtools, bcftools, python3, jupyter notebook\ -Gut microbiome WGS data with in silico mutations

python SNP_model_covid.py -i Gut_microbiome_benchmark/original_data -o Gut_microbiome_benchmark/
python SNPfilter.py -i Gut_microbiome_benchmark/
python Indelfilter.py -i Gut_microbiome_benchmark/
python SNP_model_compare.py -i Gut_microbiome_benchmark/

Point mutations detected: Gut_microbiome_benchmark/SNP_model/merge/final.txt and Gut_microbiome_benchmark/SNP_model/merge/model.sum.txt\ Indels detected: Gut_microbiome_benchmark/SNP_model/merge/indel.vcf.filtered and Gut_microbiome_benchmark/SNP_model/merge/modelindelsum.txt

-SARS-COV-2 WGS data with in silico mutations

python SNP_model_covid.py -i COVID_benchmark/original_data -fa .fasta -fq _1.fastq -o COVID_benchmark/
python SNPfilter.py -i COVID_benchmark/
python Indelfilter.py -i COVID_benchmark/
python SNP_model_compare.py -i COVID_benchmark/

-WGS data simulated with sequencing errors

python SNP_model_covid.py -i WGS_simulation_sequencingerror/original_data -fa .fasta -fq _1.fq -o WGS_simulation_sequencingerror/
python SNPfilter.py -i WGS_simulation_sequencingerror/
python Indelfilter.py -i WGS_simulation_sequencingerror/
python SNP_model_compare.py -i WGS_simulation_sequencingerror/

-MG data simulated with sequencing errors (20X)

python SNP_model_covid.py -i MG_simulation_sequencingerror/original_data -fa .fasta -fq _1.fq -o MG_simulation_sequencingerror/
python SNPfilter.py -i MG_simulation_sequencingerror/
python Indelfilter.py -i MG_simulation_sequencingerror/
python SNP_model_compare.py -i MG_simulation_sequencingerror/

-MG data simulated with sequencing errors (100X)

python SNP_model_covid.py -i MGBIG_simulation_sequencingerror/original_data -fa .fasta -fq _1.fq -o MGBIG_simulation_sequencingerror/
python SNPfilter.py -i MGBIG_simulation_sequencingerror/
python Indelfilter.py -i MGBIG_simulation_sequencingerror/
python SNP_model_compare.py -i MGBIG_simulation_sequencingerror/

-SARS-COV-2 sewage MG real data

python SNP_model_covid.py -i COVID_MGSW/original_data -fa .fasta -fq _1.fq -o COVID_MGSW/
python SNPfilter.py -i COVID_MGSW/
python Indelfilter.py -i COVID_MGSW/
python SNP_model_compare.py -i COVID_MGSW/