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
Nothing to install. This tool ships inside Liatir and runs at the version this release was tested with; it updates when Liatir updates. The tool page shows Included with Liatir and the version it is running.
On Windows it runs through WSL2 — see Windows and WSL2.
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
- Navigate to Tools → Samtools.
- Select a BAM, SAM, or CRAM file from your Data library.
- Click Run.
Liatir runs samtools flagstat on the file and displays the parsed results, keeping them in the 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.