How to prepare 454 data for dada2 denoise-pyro?

Hi,

it is my first time that I play with 454 data (yes I am that young researcher). I am interested into comparing results from other studies so therefore I extracted sff files from SRA. All samples are already demultiplexed, but they all have the barcode on the 5’ end, the primer and the targeted region.

To remove the barcode, i am planning to use the extract_barcodes.py in qiime1. However, I am confused about the primers and the use of dada2 denoise-pyro.

Should both forward and reverse primers be removed prior to use dada2? If so, do I understand well that use that method:
qiime cutadapt trim-single --i-demultiplexed-sequences demux.qza --p-front forward_primer --p-adapter reverse_primer --o-trimmed-sequences demux-trimmed.qza

or can it be done within dada2? If so, I see how I can remove the forward primer with --p-trim-left, but I am not sure how to remove the reverse primer, if it is recommended.

Thanks for your help!

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Welcome to the forum, @vca007!

Yes!

That’s the one

Since you don’t have paired-end data, no it cannot (with precision). --p-trim-left works for trimming the forward primer because you know where it would be, but length variation in your amplicon means that you don’t know quite where the reverse will be… you can use the truncation option to truncate the 3’ end of your reads and if that is removing enough then that may be all you need.

Hi,

I am currently analyzing different 454 libraries that I extracted from SRA. Each samples are therefore demultiplexed, but the barcodes and primers are still there. I figured I could use extract_barcodes.py to remove the barcode, but I am uncertain about the primers. Primers used were based on VAMPS (https://vamps.mbl.edu/resources/primers.php). For each domain, different primers but targetting the same region are used, where up to 4 different primers can be used to amplify the same region. For instance:
Bacterial v6 (454)
Forward Primers (967F)
CTAACCGANGAACCTYACC
CNACGCGAAGAACCTTANC
CAACGCGMARAACCTTACC
ATACGCGARGAACCTTACC

  • Reverse Primers (1064R)
    CGACAGCCATGCANCACCT
    CGACAACCATGCANCACCT
    CGACGGCCATGCANCACCT
    CGACGACCATGCANCACCT

These primers were pooled so they can be found in all samples.I am uncertain on how to remove these different primers simultaenously and I would greatly appreciate your help!

Thanks!

Note: if the barcodes are to the 5’ end of the forward primers, they will be removed along with the forward primer.

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Thanks Nicholas for your answers!

Here however I am still a bit confused on how to write the command line. As I wrote, 4 differently degenerated primers were used to target the same region and all four primers can be found with the same fastq file. However it seems I can only fit 1 forward and 1 reverse primer at a time with cutadapt trim-single. Is there a way to ask cutadapt to look simultaneously for 4 forward and 4 reverse primers? Thanks again!

merge the 4 into a single degenerate primer and use the --p-match-adapter-wildcards option.

Good luck!

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