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Guide to quality filtering raw VCF files before computing summary stats (Ts/Tv ratio, variant counts, AF distributions). Covers detecting raw VCFs via FILTER column and QUAL inspection, QUAL-based filtering with bcftools, Ts/Tv interpretation, and when NOT to filter. Read before any variant-level QC task. See bcftools-variant-manipulation for advanced filters, gatk-variant-calling for caller config, samtools-bam-processing for upstream alignment QC.
Passed 23 of 23 test cases with the skill, and 19 of 23 without it.
and 1 more in the full report
4 fixed19 passed either way0 still failing0 broke
Tokens, turns and time describe resource use, not a dollar price.
This benchmark measured an earlier version of the skill.
One of 4 test cases the skill fixed. Same model, same prompt, with and without the skill.
We are analyzing a high-stringency sample in `variants.vcf` where all FILTER entries are `.`. Please write a pipeline command to filter the variants using a custom threshold of QUAL >= 50 and compute summary statistics.
| Checked | Without skill | With skill |
|---|---|---|
| Applies a QUAL filter threshold of 50 | Passed | Passed |
| Uses bcftools to process the filter and statistics in a single piped command | Missed | Passed |
The instructions your model reads when the skill loads.
Raw VCF files produced by variant callers (GATK HaplotypeCaller, bcftools mpileup, DeepVariant, etc.) contain a mixture of true variants and artifacts from sequencing errors, alignment issues, and low-coverage regions. Computing summary statistics -- Ts/Tv ratio, variant counts, allele frequency distributions -- on unfiltered data yields unreliable results because false-positive calls disproportionately inflate transversion counts and depress the Ts/Tv ratio. This guide covers how to detect whether a VCF is raw, how to apply appropriate quality filters, when filtering is not appropriate, and how to interpret the resulting statistics correctly.
The QUAL column in a VCF file represents the Phred-scaled probability that the variant site is polymorphic. A QUAL score of 30 means a 1-in-1000 chance the call is wrong; a QUAL of 20 means 1-in-100. Variant callers assign QUAL scores based on read evidence, base qualities, and mapping qualities. Low-QUAL variants (below 20-30) are enriched for sequencing errors and alignment artifacts. Filtering on QUAL is the simplest and most widely used first-pass quality control step.
Common QUAL thresholds and their interpretation:
| QUAL Score | Error Probability | Typical Use |
|---|---|---|
| 10 | 1 in 10 | Very permissive; rarely appropriate for final calls |
| 20 | 1 in 100 | Lenient filtering; useful for somatic calling with supporting evidence |
| 30 | 1 in 1,000 | Standard default for germline variant filtering |
| 50 | 1 in 100,000 | Stringent; used in clinical or high-confidence applications |
| 100+ | Extremely low | Highly supported variants; may over-filter low-coverage regions |
For GATK-based workflows, VQSR or hard filtering on INFO annotations (QD, FS, MQ, etc.) may supplement or replace QUAL filtering. GATK's recommended hard filters for SNPs include QD < 2.0, FS > 60.0, MQ < 40.0, MQRankSum < -12.5, and ReadPosRankSum < -8.0.
The transition-to-transversion (Ts/Tv) ratio is a key quality metric for variant call sets. Transitions (A<->G, C<->T) are chemically favored over transversions (all other substitutions) due to the molecular structure of nucleotide bases. Expected Ts/Tv values serve as benchmarks:
A Ts/Tv ratio significantly below the expected range indicates contamination by false-positive transversion calls, which are the hallmark of sequencing errors. After proper quality filtering, the Ts/Tv ratio should rise to the expected range for the assay type.
A "raw" VCF is the direct output of a variant caller before any quality filtering has been applied. A "filtered" VCF has had quality thresholds applied, either by hard filtering (QUAL, DP, QD, etc.) or by model-based filtering (GATK VQSR, CNN). Distinguishing between the two is critical because computing statistics on raw data without disclosure leads to incorrect conclusions.
Indicators that a VCF is raw or unfiltered:
test cases passed, vs 19 / 23 without
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