cutadapt - adapter vs front

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1

Hello,

I am working with 16S sequencing data generated using 515F-806R primers.

515F: GTGCCAGCMGCCGCGGTAA
806R: GGACTACHVGGGTWTCTAAT

What is the difference between adapter and front in qiime cutadapt trim-paired?

I got the following cut adapt parameters from a colleague that generated his 16S in the same DNA core that I did using the same primers. However, I am confused about the differences between adapter vs front and I am not sure if he used the correct sequence for each parameter.

qiime cutadapt trim-paired\
   --i-demultiplexed-sequences paired-end-demux.qza\
   --p-cores 10\
   --p-adapter-f ATTAGAWACCCBDGTAGTCC\
   --p-front-f GTGCCAGCMGCCGCGGTAA\
   --p-front-r GGACTACHVGGGTWTCTAAT\                                                                                                                      
   --p-adapter-r TTACCGCGGCKGCTGGCAC\
   --o-trimmed-sequences trimmed-paired-end-demux.qza

In his code, the 515F sequence was entered in --p-front-f, and 806R sequence in --p-front-r. From what I could gather, an index sequence primer (ATTAGAWACCCBDGTAGTCC) was entered in --p-adapter-f and 515 reverse sequence (TTACCGCGGCKGCTGGCAC) was used for --p-adapter-r .

I found it odd to have an index sequencing primer specified on --p-adapter-f and then the 515 reverse on '--p-adapter-r'. Is this correct?

From what I've read, I am with the impression that there is no real difference between adapter and front, and that they are just two similar options to remove different primers from the 5' or 3' ends. Am I too far off?

Thanks for your attention. I’m looking forward to your reply.

2

@JoaoGabrielMoraes, I feel like you're asking two different questions here, and I can only answer the first one confidently :sweat_smile:

1. I'm feeling good here...

This is covered in the docs:
adapter is the "Sequence of an adapter ligated to the 3' end" of whichever read is specified in the parameter (e.g. --p-adapter-f is the sequence of an adapter ligated to the 3` end of the forward read)

front is the "Sequence of an adapter ligated to the 5' end" of whichever read is specified in the parameter.

The cutadapt docs go into depth about the various ways these and other parameters can be used to describe different barcode/primer placements.

2. Please take this attempt at an answer with a grain of salt.

I suspect your colleague went about things the right way, but I'm not sure my understanding of how forward and reverse reads work is correct. I'm imagining a matched pair of reads looks like this...
(5') 515primer->fwd-sequence->index (3') ...... (3') index<-rev-sequence<-806primer (5')

... so both primers get assigned to front parameters, while the 3' ends are on the "inside" and are assigned to adapter.

Does this line up with your understanding of the sequencing process? Does this make sense?

Best,
Chris :leopard:

4

Hi @ChrisKeefe,

Thank you very much for clarifying this! My colleague's code now makes sense to me.

--p-adapter-f and --p-adapter-r are actually the reverse complement of 515F and 806R.

I was just worried about having to remove additional adaptors from Illumina sequencing (e.g. i5 and i7 adaptors). However, the Illumina adaptors are closer to the ends than the primers so they are trimmed off when trimming out the primers.

Thanks again for your help!!

Best, Joao