Samtools

Samtools is the standard toolkit for working with sequence alignment data. Liatir uses the flagstat subcommand to produce a quick summary of alignment statistics from BAM, SAM, or CRAM files.

Details

Property	Value
Type	Native tool
Binary	samtools
Subcommand	flagstat

Installation

Samtools must be installed and available in your system PATH.

macOS (Homebrew)

brew install samtools

Ubuntu/Debian

sudo apt install samtools

conda

conda install -c bioconda samtools

Liatir checks for samtools on page load. If it is not found, the dependency card shows these same instructions.

Accepted inputs

Extension	Description
.bam	Binary alignment map (the standard format)
.sam	Text alignment map
.cram	Compressed, reference-based alignment format

BAM is the most common format. SAM files are large (text) and uncommon in practice; most pipelines write BAM directly.

BAM indexing

samtools flagstat reads the entire file sequentially — it does not need a .bai index. Other samtools operations (like view -r for region queries) do require an index.

Running Samtools flagstat

1. Navigate to Tools → Samtools.
2. Select a BAM, SAM, or CRAM file from your Data library.
3. Click Run.

The run spawns samtools flagstat <path> and captures stdout. Results are parsed and stored in run history.

Output metrics

samtools flagstat counts reads in each category defined by the SAM bitwise FLAG field. Each count is reported twice: reads that pass QC filters and reads that fail them (the latter shown in parentheses).

Metric	FLAG bits	Description
Total reads	—	All records in the file (QC-passed + QC-failed)
Mapped	0x4 unset	Reads with at least one reported alignment
Mapped %	—	Mapped / total × 100
Duplicates	0x400	Reads marked as optical or PCR duplicate
Properly paired	0x2	Both mates aligned, within expected distance and orientation
Secondary	0x100	Alternative alignments for multi-mapping reads
Supplementary	0x800	Chimeric alignments (e.g., from structural variant evidence)
Paired in sequencing	0x1	Reads from paired-end library
Read 1	0x40	First mate in a pair
Read 2	0x80	Second mate in a pair

Interpreting results

Metric	Good range	Concern
Mapped %	≥ 95% (WGS human)	< 80% may indicate wrong reference, contamination, or damaged library
Properly paired %	Close to mapping rate	Large gap may indicate structural rearrangements or library quality issues
Duplicate %	< 20% (WGS)	> 40% suggests over-amplification; may bias variant calling and coverage
Secondary	Low (< 5%)	High secondary counts mean many reads map to multiple locations

Duplicate rate context

Duplicate percentages are expected to be higher for:

• Amplicon sequencing (by design)
• Very low-input libraries
• High-depth targeted panels

For whole-genome or whole-exome, 10–25% is typical. Above 40% is worth investigating.