16S CCS without primers: how to skip --p-front/--p-adapter in denoise-ccs?

User Support
1 
Hi everyone,

I’m currently analyzing full-length 16S rRNA PacBio CCS data with QIIME 2 (version: 2024.2). The data I received from the sequencing provider were already delivered without any  primer sequences.I now want to run:

qiime dada2 denoise-ccs \
  --i-demultiplexed-seqs reads_qza/raw_reads.qza \
  --p-min-len 1000 \
  --p-max-len 1600 \
  --p-front AGRGTTYGATYMTGGCTCAG \
  --p-adapter RGYTACCTTGTTACGACTT \
  --p-n-threads $NCORES \
  --o-table dada2_output/table.qza \
  --o-representative-sequences dada2_output/representative_sequences.qza \
  --o-denoising-stats dada2_output/stats.qza \
  --verbose

The problem is that denoise-ccs treats --p-front and --p-adapter as required parameters; if I omit them, the command throws an error. How should I modify the command (or the parameter values) to proceed?

Thank you!

Zhen
2

Hi @Zxz-xiao ,

Welcome to the forum! I haven’t used this particular DADA2 tool before, but could you tell me what version of QIIME2 you are using? According to these docs and these docs, it looks like only the --front parameter is required for this command. Do you have the sequence of the primer used at the 5' end in your single-end read sequencing? It’s possible that the sequencer removed the machine adapters but not the primers.

Unless someone knows a way to bypass this, feel free to chime in. From what I understand, if a read doesn’t contain the inputed sequence, it will be discarded, so I don’t think you can just supply a “dummy” sequence to bypass it as this will probably result in the majourity of your sequences being throw away.

If that’s the case, you may need to contact the sequencing facility to provide the raw data, which shouldn’t be difficult (and in general, it’s better to work with the raw data anyway).

Hope that helps.

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3

Thank you for the helpful reply. The version I’m using is 2024.2 and I will request the raw data from the sequencing provider to do this.

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4

@Zxz-xiao Good plan. I would also try to use an up to date version of Qiime2, then you can just supply the front adapter.

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