Flag reference notes

Run trim_galore --help for the complete list of options with one-line descriptions and default values. This section focuses on flag interactions and scenarios that benefit from more context than the terse --help output.

Cross-flag interactions

A few combinations are worth knowing about:

• --small_rna lowers the --length default to 18 bp (from 20) and auto-sets --adapter2 to the Illumina small RNA 5' adapter (GATCGTCGGACT) for paired-end data, unless the user provides their own --adapter2 value. The same holds for --bgiseq, which auto-sets the BGISEQ-500 Read 2 adapter.
• --rrbs in paired-end directional mode auto-sets --clip_R2 2 to mask the 2 bp end-repair bias at the start of Read 2, unless the user provides their own --clip_R2 value. --non_directional intentionally skips this auto-clip.
• --library <preset> expands into the four clip flags for that kit and nothing else (see Library presets). An explicit --clip_R1 / --clip_R2 / --three_prime_clip_R1 / --three_prime_clip_R2 wins over the preset value it displaces, and both numbers are logged. A preset that supplies --clip_R2 also suppresses the --rrbs auto-clip described above, since the value is already set. It is refused with --hardtrim5, --hardtrim3, --clock and --implicon, which do not honour the clip flags.
• --clip_R1 / --clip_R2 / --three_prime_clip_R1 / --three_prime_clip_R2 warn and are ignored where the run cannot honour them: the two Read 2 flags on a single-end run, and all four under --hardtrim5, --hardtrim3, --clock and --implicon, which do their own fixed clipping. The text trimming report lists only the clipping that applied, so a single-end report names Read 1 alone; the JSON report records the values as requested, alongside a mode key saying which run type they were requested for.
• -a2 / --adapter2 warns and is ignored in the same four shapes, for two different reasons: under --hardtrim5, --hardtrim3, --clock and --implicon, because those modes perform no adapter trimming at all, and on any single-end run, because there is no Read 2 to trim. The warning names which of the four applied: WARNING: -a2/--adapter2 was given but is not used in this mode (<reason>). Ignoring. Note that in those four shapes the value is not parsed either, so dropping --paired turns a malformed -a2 from a hard error into silence, and a valid one from applied into ignored.
• --paired + --length discards the whole read pair unless both reads pass the length cutoff. To rescue the surviving read when only one becomes too short, add --retain_unpaired; the per-side cutoff is governed by --length_1/--length_2 (default 35 bp each).
• --trim-n is suppressed under --rrbs (matches Perl v0.6.x; N-trimming interacts poorly with RRBS end-repair masking).
• --discard_untrimmed keeps only reads where at least one adapter match was found. For paired-end, the pair is discarded only if neither read had an adapter.
• --fastqc (and --fastqc_args, which implies it) is rejected on the four specialty modes and on --paired given a single interleaved uBAM with FASTQ output. Those paths write no report, so the flag fails loudly rather than being ignored; for the last one, adding --output-format ubam does produce a report. See FastQC.
• --poly_g is auto-enabled when the data looks like it came from a 2-colour instrument (sequence-based detection on trailing G-runs). Use --no_poly_g to force-disable, or --poly_g to force-enable. It is independent of --nextseq (which is quality-score-based).

RRBS-specific guidance

For library kits where --rrbs should not be used (Tecan Ovation RRBS Methyl-Seq, MseI-digested libraries), see When NOT to use --rrbs.

Adapter specification recap

See Multiple adapter sequences for the three equivalent syntaxes (repeatable -a/-a2, embedded-string form, and file:adapters.fa). Supplementary notes:

• Single-base expansion -a A{N} / -a2 A{N} repeats the base N times, matching Perl v0.6.x syntax.
• Adapter auto-detection runs per pair in v2.x (Perl ran it once per invocation), so a shell glob trim_galore --paired *fastq.gz correctly handles multiple samples with different library types or 2-colour/4-colour chemistries.
• --consider_already_trimmed <INT> suppresses adapter trimming entirely when no auto-detect probe exceeds that count (quality trimming still runs). Useful for feeding already-trimmed data through Trim Galore for QC reporting without over-trimming.

Perl-era flag forms (still supported)

To keep v0.6.x scripts working unchanged, a small pre-parse hook recognises these Perl-era spellings and rewrites them to the Clap-friendly long-alias forms:

• -r1 / -r2 rewrites to --r1 / --r2
• -a2 rewrites to --a2
• -a " SEQ1 -a SEQ2" (embedded-string form)

Plus A{N} brace expansion in -a / -a2. See the migration guide for the full picture.

Where to find what

You want to…	Look here
Tune Phred quality trimming	Quality trimming
Specify or override adapters	Adapter trimming
Rescue under-length pairs	Paired-end data
Set length and max-N cutoffs	Length filtering
Read a trimming report	Trimming reports
Trim RRBS / bisulfite libraries	RRBS mode and Bisulfite & RRBS
Use a specialty UMI mode	Mouse Epigenetic Clock and IMPLICON