📋 1-Column Reference Guide VCF • BAM • BED • GFF3 Samtools • Bcftools • Bedtools • Seqkit

The Bioinformatician's Command Cookbook & Format Reference

High-yield command-line one-liners, CLI tool syntax cards, and comprehensive field-by-field specifications for core genomic formats. Arranged in a streamlined 1-column layout so commands and explanations sit cleanly on a single line.

Jump to: 🧪 In-Browser WASM Terminal ⚡ Shell One-Liners 🛠️ Samtools & Bcftools 📐 Bedtools Intervals ✂️ Seqkit Utilities 📄 Format Specifications

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FASTQ

Count Total Reads (Gzipped)

— Divides raw line sum by 4 in a streaming pipeline without disk decompression.

zcat reads.fq.gz | awk '{s++}END{print s/4}'

FASTQ

Convert to FASTA Format

— Substitutes '@' with '>' on headers and preserves nucleotide rows while discarding qualities.

zcat reads.fq.gz | paste - - - - | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > out.fa

FASTQ

Filter Reads by Sequence Motif

— Isolates 4-line records bearing a specific primer or adapter oligomer.

zcat reads.fq.gz | grep -B 1 -A 2 --no-group-separator "GATTACA" | gzip > matched_reads.fq.gz

BAM

Retain Mapped High-Quality Alignments

— Excludes unmapped segments (-F 4) and enforces MAPQ ≥ 30 (-q 30).

samtools view -b -F 4 -q 30 in.bam > filtered.bam

BAM

Count Alignments per Chromosome

— Rapid summary retrieved from the .bai index without reading raw records.

samtools idxstats in.bam | cut -f 1,3 | head -n 25

BAM

Quantify PCR & Optical Duplicates

— Leverages bitwise flag 1024 to report total redundant read pairs.

samtools view -c -f 1024 in.bam

VCF

Filter for PASS & High-Frequency SNVs

— Extracts confident variants meeting all quality criteria with Allele Frequency > 5%.

bcftools view -f 'PASS' -i 'TYPE="snp" && INFO/AF > 0.05' in.vcf.gz -Oz -o filtered.vcf.gz

VCF

Calculate Transition / Transversion (Ti/Tv)

— Expected human WGS baseline is ~2.0–2.1; exome targets reach ~2.8–3.0.

bcftools stats in.vcf.gz | grep "^TSTV" | cut -f 5

VCF

Sort Coordinates, Bgzip & Tabix Index

— Orders raw callsets into standard genomic sequence and generates .tbi lookup.

bcftools sort in.vcf -Oz -o sorted.vcf.gz && tabix -p vcf sorted.vcf.gz

🛠️ Samtools & Bcftools Syntax Cards

v1.19+ Standards

SAMTOOLS

Coordinate-Sort Alignments to BAM

— Spins 8 CPU worker threads (-@ 8) for accelerated binary compression.

samtools sort -@ 8 -o out.bam in.sam

SAMTOOLS

Index Binary Alignment File

— Builds the .bai index necessary for random locus queries and IGV navigation.

samtools index -@ 4 in.bam

SAMTOOLS

Flagstat Alignment Summary

— Reports total segments, primary mappings, proper pairs, and duplicate percentages.

samtools flagstat in.bam > stats.txt

SAMTOOLS

Per-Base Depth across Target Regions

— -a outputs all loci including zero-coverage; -b restricts to intervals.

samtools depth -a -b targets.bed in.bam > depth.tsv

SAMTOOLS

Slice Genomic Window by Coordinates

— Extracts reads overlapping a target interval directly from indexed BAM.

samtools view -b in.bam "chr17:41196312-41277500" > brca1_reads.bam

BCFTOOLS

Normalize & Split Multiallelic Sites

— Left-aligns indels and partitions multi-ALT records into individual biallelic rows.

bcftools norm -m -any -f ref.fa in.vcf.gz -Oz -o norm.vcf.gz

BCFTOOLS

Export Multi-Sample Genotypes to TSV

— Square brackets [...] automatically iterate across all sample columns.

bcftools query -f '%CHROM\t%POS\t%REF\t%ALT[\t%GT]\n' in.vcf.gz > genotypes.tsv

BCFTOOLS

Filter Variants by Quality & Depth

— Retains PASS calls having Phred score ≥ 30 and DP ≥ 15.

bcftools view -i 'FILTER="PASS" && QUAL>=30 && DP>=15' in.vcf.gz -Oz -o filtered.vcf.gz

📐 Bedtools Interval Arithmetic

Genomic Arithmetic

BEDTOOLS

Intersect Overlapping Features

— Finds shared genomic regions between feature sets A and B (e.g. ChIP peaks in Promoters).

bedtools intersect -a peaks.bed -b promoters.bed -wa -wb > overlaps.bed

BEDTOOLS

Merge Contiguous Intervals

— Collapses overlapping or adjacent spans into unified genomic segments.

sort -k1,1 -k2,2n in.bed | bedtools merge -i - > merged.bed

BEDTOOLS

Subtract Excluded Regions

— Strips regions in set B from features in A (e.g. removing repeats from exons).

bedtools subtract -a exons.bed -b repeats.bed > non_repeat_exons.bed

BEDTOOLS

Extract Reference Sequences

— Pulls FASTA sequence corresponding to coordinate intervals from reference.

bedtools getfasta -fi hg38.fa -bed peaks.bed -fo peaks.fa

BEDTOOLS

Find Nearest Feature

— Identifies non-overlapping neighbor in B for each interval in A, returning distance (-d).

bedtools closest -a variants.bed -b genes.bed -d > closest_genes.bed

BEDTOOLS

Calculate Breadth & Depth

— Summarizes mapped coverage, read counts, and percentage across intervals.

bedtools coverage -a targets.bed -b sample.bam > coverage_summary.bed

✂️ Seqkit Sequence Utilities

Ultra-Fast Go Binary

SEQKIT

Summary Statistics (N50, Q20, Q30)

— Generates record counts, length ranges, N50 assembly metric, and Phred distributions.

seqkit stats -a -j 4 *.fastq.gz

SEQKIT

Filter Records by Header List

— Pulls matching FASTA/FASTQ records using a plain text file of accession names.

seqkit grep -f id_list.txt in.fasta -o filtered.fasta

SEQKIT

Extract Coordinate Sub-Sequence

— Slices exact nucleotide windows (e.g. 1 to 5000 bp) without indexing overhead.

seqkit subseq -r 1:5000 in.fasta -o promoter_window.fa

SEQKIT

Reverse Complement & Linearize

— -r -p generates reverse complements; -w 0 converts wrapped entries to single-line.

seqkit seq -r -p -w 0 in.fasta -o revcomp.fasta

SEQKIT

Deduplicate Records by Content

— -s identifies duplicate sequences; -d redirects redundant entries to an audit file.

seqkit rmdup -s -i in.fasta -o dedup.fasta -d duplicates.fasta

SEQKIT

Order Contigs by Length

— -l orders by size; -r sorts in descending sequence for assembly inspection.

seqkit sort -l -r in.fasta -o sorted_by_length.fasta

📄 File Format Specifications

Community Standards

VCF 4.2 / 4.3 Mandatory Columns

Every VCF features 8 mandatory tab-separated fields, followed by an optional 9th column (FORMAT) and sample calls (10+).

Col #	Field Name	Type / Convention	Description
1	CHROM	String	Chromosome identifier (e.g. `chr17` or `17`).
2	POS	Integer (1-based)	Starting coordinate of the first base in the REF allele.
3	ID	String	Accession identifier (e.g. dbSNP rsID `rs121913529` or `.` if unassigned).
4	REF	String [A,C,G,T,N]	Nucleotide bases present in the source genome build.
ALT	Comma-separated	Non-reference alleles observed in the sequenced sample.
6	QUAL	Float (Phred)	`-10 * log10(P(error))` for the alternate call. Score 30 represents 99.9% confidence.
7	FILTER	String	`PASS` when meeting all quality criteria; otherwise lists failed metric names.
8	INFO	Key-Value (Semicolon)	Site-level annotations (e.g. `DP=120;AF=0.45;AN=2;AC=1`).

Essential Sample FORMAT Sub-Tags (Col 9+):

GT Genotype Call: 0/0 ref hom, 0/1 het, 1/1 alt hom, 0|1 phased chromosome configuration

AD Allelic Depths: Unfiltered count of passing reads for REF and each ALT allele (e.g. 32,18)

DP Sample Read Depth: Filtered coverage sum evaluated across this specific locus (e.g. 50)

GQ Call Confidence: Phred metric (0–99) evaluating certainty in the assigned GT

PL Normalized Likelihoods: Phred calculations across possible genotypes (0 indicates the most probable state, e.g. 0,45,255)

SAM / BAM 11 Mandatory Columns

The Sequence Alignment/Map standard requires 11 mandatory tab-delimited columns per aligned segment.

Col #	Field Name	Type	Description
1	QNAME	String	Query template / read identifier (e.g. `A00123:14:H7K5MDSXX:1:1101:1000:2050`).
2	FLAG	Bitwise Int	Binary indicators (e.g. 99 = read paired, proper pair, mate reverse strand, first in pair).
3	RNAME	String	Reference chromosome where segment aligns (e.g. `chr1`).
4	POS	1-Based Pos	Leftmost coordinate of the first aligned base.
5	MAPQ	Integer (Phred)	Confidence metric: `-10 * log10(P(mapping error))`. 60 represents highest certainty.
CIGAR	String	Alignment operations string (e.g. `100M` match or `15S85M` soft-clipping).
RNEXT	String	Chromosome of the mate pair (`=` if identical).
8	PNEXT	Integer	1-based position of the mate read.
9	TLEN	Integer	Observed template span / insert size between paired reads.
10	SEQ	String [A,C,G,T,N]	Nucleotide sequence represented on reference strand.
11	QUAL	String (ASCII)	Phred score ASCII characters matching 1:1 with SEQ bases.

CIGAR String Operators (Column 6):

M Match or Mismatch: Bases aligned to the reference genome; denotes sequence identity or single-nucleotide variant.

I Insertion: Novel bases present in the read that are absent from the reference assembly.

D Deletion: Bases missing in the read segment that exist in the reference genome.

N Intron Skip: Spliced junction spanning mRNA exons in RNA-seq alignments.

S Soft Clipping: Terminal bases (adapter, primer, low quality) excluded from alignment but kept in SEQ.

H Hard Clipping: Excluded bases fully discarded and omitted from the SEQ field.

Example: 15S85M1200N50M

→ 15 bp soft-clipped adapter/primer + 85 bp exon alignment + 1,200 bp spliced intron + 50 bp downstream exon alignment.

BED Format Standard 3, 6 & 12 Columns

The Browser Extensible Data standard utilizes 0-based, half-open coordinates [chromStart, chromEnd).

Col #	Field Name	Format Tier	Description
1	chrom	BED3+	Chromosome name (e.g. `chr7`).
2	chromStart	BED3+	0-based starting coordinate of the feature.
3	chromEnd	BED3+	1-based boundary (half-open, length = end - start).
4	name	BED6+	Feature identifier or symbol (e.g. `EGFR_exon1`).
5	score	BED6+	Quality ranking integer between 0 and 1000.
6	strand	BED6+	Directional orientation: `+` (forward), `-` (reverse), or `.` (unassigned).
7–12	thickStart..blockStarts	BED12	Defines coding boundaries (thickStart/End), RGB color, exon block count, block sizes, and relative block starts.