RRBS mode

Trim galore! also has an --rrbs option for DNA material that was digested with the restriction enzyme MspI. In this mode, Trim Galore identifies sequences that were adapter-trimmed and removes another 2 bp from the 3' end of Read 1, and for paired-end libraries also the first 2 bp of Read 2 (which is equally affected by the fill-in procedure). This is to avoid that the filled-in cytosine position close to the second MspI site in a sequence is used for methylation calls. Sequences which were merely trimmed because of poor quality will not be shortened any further.

# Single-end RRBS
trim_galore --rrbs sample.fq.gz

# Paired-end RRBS (auto-sets --clip_R2 2)
trim_galore --rrbs --paired sample_R1.fq.gz sample_R2.fq.gz

For the biology behind RRBS (strand types, fill-in artifacts, and why trimming is needed), see the Bisulfite & RRBS guide.

Non-directional mode

Trim Galore also has a --non_directional option, which will screen adapter-trimmed sequences for the presence of either CAA or CGA at the start of sequences and clip off the first 2 bases if found. If CAA or CGA are found at the start, no bases will be trimmed off from the 3' end even if the sequence had some contaminating adapter sequence removed (in this case the sequence read likely originated from either the CTOT or CTOB strand; refer to the RRBS guide for the meaning of CTOT and CTOB strands).

trim_galore --rrbs --non_directional --paired sample_R1.fq.gz sample_R2.fq.gz

--non_directional intentionally skips the auto --clip_R2 2 that directional --rrbs applies, since the 5' bias on Read 2 is handled by the start-clip logic instead.

What --rrbs changes vs default mode

	Default	--rrbs
Quality trim	Yes	Yes
Adapter trim	Yes	Yes
Extra 2 bp clip on adapter-trimmed Read 1 (3' end)	No	Yes
Auto --clip_R2 2 (paired-end)	No	Yes (directional only)
--trim-n	If passed	Suppressed (matches Perl v0.6.x)

--trim-n is suppressed under --rrbs because N-trimming interacts poorly with RRBS end-repair masking. This restores byte-identical output with Perl v0.6.x for users who combine both flags.

When NOT to use --rrbs

Two cases come up regularly.

Tecan Ovation RRBS Methyl-Seq kit (with or without TrueMethyl oxBS 1-16): the Tecan kit attaches a varying number of nucleotides (0-3) after each MspI site, so standard --rrbs 2 bp 3'-trimming over-trims. Run Trim Galore without --rrbs and handle the fill-in via Tecan's subsequent diversity-trimming step (see their manual).

MseI-digested RRBS libraries: if your DNA was digested with MseI (recognition motif TTAA) instead of MspI, you do not need --rrbs or --non_directional. Virtually all reads should start with TAA, and the end-repair of TAA-restricted sites does not involve cytosines, so no special treatment is required. Just run Trim Galore in the default (non-RRBS) mode.

Related flags

Flag	Purpose
--rrbs	Enable RRBS mode (MspI fill-in handling).
--non_directional	Strip CAA/CGA at start of CTOT/CTOB-derived reads.
--clip_R2 N	Override the auto 2 bp clip on Read 2 (RRBS paired-end).
--three_prime_clip_R1 N / --three_prime_clip_R2 N	Manual 3' clip on either side.