cutadapt doesn't trim all primers

Question

[fladveronika]

Hi together, I'm new to the analysis of amplicon sequencing reads with dada2 and cutadapt in R. I'm following this tutorial (worked with the test data set) integrating my own data: https://benjjneb.github.io/dada2/ITS_workflow.html After the step, in which cutadapt trims the primers from my sequences, it doesn't detect the forward primer in all sequences. I assume that I would have to change some settings so that the leftover primer will also be found. Here is what I do:

# Loading cutadapt in R
cutadapt <- "~pathto directory"
system2(cutadapt, args = "--version")
# Define output files for cutadapt
path.cut <- file.path(path16S, "cutadapt")
if(!dir.exists(path.cut)) dir.create(path.cut)
fnFs.cut <- file.path(path.cut, basename(fnFs))
fnRs.cut <- file.path(path.cut, basename(fnRs))
# Define primers to search for
FWD.RC <- dada2:::rc(FWDPro341F)
REV.RC <- dada2:::rc(REVPro805R)
# Trim FWD and the reverse-complement of REV off of R1 (forward reads)
R1.flags <- paste("-g", FWDPro341F, "-a", REV.RC)
# Trim REV and the reverse-complement of FWD off of R2 (reverse reads)
R2.flags <- paste("-G", REVPro805R, "-A", FWD.RC)
# Run Cutadapt
for(i in seq_along(fnFs)) {
  system2(cutadapt, args = c(R1.flags, R2.flags, "-n", 2, "-o", fnFs.cut[i], "-p", fnRs.cut[i], fnFs.filtN[i], fnRs.filtN[i]))}
Summarized output: 
=== Summary ===

Total read pairs processed:             18,890
  Read 1 with adapter:                  18,502 (97.9%)
  Read 2 with adapter:                  18,739 (99.2%)
Pairs written (passing filters):        18,890 (100.0%)

Total basepairs processed:    11,334,000 bp
  Read 1:     5,667,000 bp
  Read 2:     5,667,000 bp
Total written (filtered):     10,521,217 bp (92.8%)
  Read 1:     5,297,096 bp
  Read 2:     5,224,121 bp

# Check for primers in the first cutadapted sample
rbind(FWD.ForwardReads = sapply(FWD341.orients, primerHits, fn = fnFs.cut[[1]]), 
      FWD.ReverseReads = sapply(FWD341.orients, primerHits, fn = fnRs.cut[[1]]), 
      REV.ForwardReads = sapply(REV805.orients, primerHits, fn = fnFs.cut[[1]]), 
      REV.ReverseReads = sapply(REV805.orients, primerHits, fn = fnRs.cut[[1]]))
After running this, I get the following output:
                 Forward Complement Reverse RevComp
FWD.ForwardReads       1          0       0       0
FWD.ReverseReads       0          0       0       0
REV.ForwardReads       0          0       0       0
REV.ReverseReads       0          0       0       0

So obviously I have a problem, by googling I got the hint I must change the errors/mismatches within the primer sequence, but I can't find how to do this. Any suggestions? Best regards, Veronika Flad

[Arup Ghosh]

I didn't find any reference to FWDPro341F and REVPro805R in your code. Check the cutadapt guide primer trimming guide for more options and tweaks.

https://cutadapt.readthedocs.io/en/stable/recipes.html#trimming-amplicon-primers-from-both-ends-of-paired-end-reads