Poly-G and Poly-A trimming

v2 ships with two built-in poly-X trimmers that the Perl version did not have: --poly_g for 2-colour-instrument no-signal G-runs, and --poly_a for poly-A-tailed mRNA-seq libraries.

Poly-G on 2-colour instruments

On 2-colour Illumina instruments (NextSeq, NovaSeq, NovaSeq X), no signal is encoded as a high-quality G. This produces spurious G-runs at the 3' end of reads with little signal. They look like high-quality bases to the basecaller but represent dark cycles, not actual G calls.

--poly_g scans the 3' end of Read 1 (and the 5' end of Read 2, where the reverse complement reads as poly-C) for these runs and trims them. It is auto-enabled when the data looks 2-colour, based on sequence detection of trailing G-runs.

# Auto-detection runs by default. No flag needed.
trim_galore input.fq.gz

# Force-enable
trim_galore --poly_g input.fq.gz

# Force-disable
trim_galore --no_poly_g input.fq.gz

--poly_g is independent of --nextseq and --2colour:

• --2colour N (alias --nextseq N) is quality-score based and replaces -q. It treats trailing Gs as low-quality during quality trimming.
• --poly_g is sequence-based and runs after quality trimming. It looks for actual G-runs, regardless of their quality scores.

You can use them together. They target overlapping artifacts.

Poly-A for mRNA-seq libraries

--poly_a trims poly-A tails from the 3' end of reads. Use this for mRNA-seq and poly-A-selected RNA-seq libraries, where the poly-A tail can survive into the read.

trim_galore --poly_a input.fq.gz

In paired-end mode, the corresponding poly-T is removed from the 5' end of the mate read.

--poly_a is opt-in. It is not auto-detected. Pass it when you know your library is poly-A selected.

When to use which

Library type	Recommendation
2-colour instrument (NextSeq, NovaSeq, NovaSeq X)	Leave --poly_g auto-detection on. Add --2colour 20 if you also want quality-score-based handling.
4-colour instrument (HiSeq, MiSeq)	No poly-G needed. --poly_g will not auto-trigger.
Poly-A-selected mRNA-seq	Add --poly_a.
Total RNA / ribodepleted	Skip --poly_a. There are no poly-A tails to trim.