I am trying to use bbduk to do adapter and quality trimming and then use ftl=12.
When does the force trimming happen - I know that adapter trimming happens before quality trimming
My question: what's the order of operations if I use
bbduk.sh in=read.fq out=clean.fq ref=adapters.fa ftl=12 minlength=35 trimq=30 ktrim=r qtrim=rl k=23 mink=11 hdist=1
I am assuming that adapter trimming happens first, then quality trimming and then positional trimming -
Context: I am trying to do this based on Lexogen recommendation for Quantseq FWD: https://www.lexogen.com/quantseq-3mrna-sequencing/#quantseqfaq
What sequence should be trimmed: The reads should be trimmed to remove adapter sequences, poly(A) / poly(T) sequences, and low quality nucleotides. Reads that are too short (i.e., <20 nt) or have generally low quality scores should be removed from the set. As second strand synthesis is based on random priming, there may be a higher proportion of errors at the first nucleotides of the insert due to non-specific hybridization of the random primer to the cDNA template. For QuantSeq FWD data we therefore recommend using an aligner that can perform soft-clipping of the read ends (e.g., STAR aligner) during alignment, or increasing the number of allowed mismatches to 14. Alternatively, trimming the first 12 nt of Read 1 can be performed prior to alignment when using a more stringent aligner (e.g., HISAT2). While trimming the read can decrease the number of reads of suitable length for alignment, the absolute number of mapping reads may increase due to the improved read quality.
Thanks in advance