Hello qiime users,
I apologize if there is some missing, wrong or confused information.
I am using qiime2-2021.4 on linux
I am processing COI data from a 2x250 Illumina MiSeq run. Samples were never multiplexed, so I proceeded after importing with primer removal using cutadapt. For the run was used Leray (2013) primers that should have a length of 313 bp.
Below is my sequence quality profile and the parameter that seems to retain the most sequences at each step, but also reduces the sequence length to 234 bp.
Do you have any suggestions for DADA2 filtering/merging/denoising step for fixing the problem?
Can you see any problem with the sequencing run or library preparation? it is necessary to rerun that samples?
I used these commands
qiime cutadapt trim-paired --i-demultiplexed-sequences paired-end-COI.qza --p-cores 8 --p-front-f GGWACWGGWTGAACWGTWTAYCCYCC --p-front-r TAAACTTCAGGGTGACCAAAAAATCA --o-trimmed-sequences COI-trimmed.qza --verbose
qiime dada2 denoise-paired --p-n-threads 8 --i-demultiplexed-seqs COI-trimmed.qza --p-trunc-len-f 205 --p-trunc-len-r 179 --o-table table-dada.qza --o-representative-sequences rep-seqs-dada.qza --o-denoising-stats denoising-stats-dada.qza --verbose
The last sample (T) is a mock community of known species that always gives no merged sequences.
I’ve tried adjusting the parameters (varying truncation lengths, increasing --p-max-ee-f and --p-max-ee-r numbers up to 15, trimming off 36 bp)
Are these good data to be processed?
Could you suggest me something for improving my results? Is this the best I can expect from my data?
Thank you in advance for your reply. I hope that can be useful for all qiime2 users.